Processes for preparing atr inhibitors

ABSTRACT

wherein the variables are as defined herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S.Non-provisional patent application Ser. No. 13/631,759, filed Sep. 28,2012, which claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 61/541,865, filed on Sep. 30, 2011, thecontents of which are incorporated by reference.

BACKGROUND OF THE INVENTION

ATR (“ATM and Rad3 related”) kinase is a protein kinase involved incellular responses to DNA damage. ATR kinase acts with ATM (“ataxiatelangiectasia mutated”) kinase and many other proteins to regulate acell's response to DNA damage, commonly referred to as the DNA DamageResponse (“DDR”). The DDR stimulates DNA repair, promotes survival andstalls cell cycle progression by activating cell cycle checkpoints,which provide time for repair. Without the DDR, cells are much moresensitive to DNA damage and readily die from DNA lesions induced byendogenous cellular processes such as DNA replication or exogenous DNAdamaging agents commonly used in cancer therapy.

Healthy cells can rely on a host of different proteins for DNA repairincluding the DDR kinase ATR. In some cases these proteins cancompensate for one another by activating functionally redundant DNArepair processes. On the contrary, many cancer cells harbour defects insome of their DNA repair processes, such as ATM signaling, and thereforedisplay a greater reliance on their remaining intact DNA repair proteinswhich include ATR.

In addition, many cancer cells express activated oncogenes or lack keytumour suppressors, and this can make these cancer cells prone todysregulated phases of DNA replication which in turn cause DNA damage.ATR has been implicated as a critical component of the DDR in responseto disrupted DNA replication. As a result, these cancer cells are moredependent on ATR activity for survival than healthy cells. Accordingly,ATR inhibitors may be useful for cancer treatment, either used alone orin combination with DNA damaging agents, because they shut down a DNArepair mechanism that is more important for cellular survival in manycancer cells than in healthy normal cells.

In fact, disruption of ATR function (e.g. by gene deletion) has beenshown to promote cancer cell death both in the absence and presence ofDNA damaging agents. This suggests that ATR inhibitors may be effectiveboth as single agents and as potent sensitizers to radiotherapy orgenotoxic chemotherapy.

For all of these reasons, there is a need for the development of potentand selective ATR inhibitors for the treatment of cancer, either assingle agents or as combination therapies with radiotherapy or genotoxicchemotherapy. Furthermore, it would be desirable to have a syntheticroute to ATR inhibitors that is amenable to large-scale synthesis andimproves upon currently known methods.

ATR peptide can be expressed and isolated using a variety of methodsknown in the literature (see e.g., Ünsal-Kaçmaz et al, PNAS 99: 10, pp6673-6678, May 14, 2002; see also Kumagai et al. Cell 124, pp 943-955,Mar. 10, 2006; Unsal-Kacmaz et al. Molecular and Cellular Biology,February 2004, p 1292-1300; and Hall-Jackson et al. Oncogene 1999, 18,6707-6713).

DESCRIPTION OF THE FIGURES

FIG. 1 a: XRPD Compound I-2 free base

FIG. 2a : TGA Compound I-2 free base

FIG. 3a : DSC Compound I-2 free base

FIG. 4a : ORTEP plot of the asymmetric unit of the Compound I-2 freeform single crystal structure

FIG. 1 b: XRPD Compound I-2.HCl

FIG. 2b : TGA Compound I-2.HCl

FIG. 3b : DSC Compound I-2.HCl

FIG. 4b : ORTEP plot of the asymmetric unit of the Compound I-2.HClanhydrous structure.

FIG. 1 c: XRPD Compound I-2.2HCl

FIG. 2c : TGA Compound I-2.2HCl

FIG. 3c : DSC Compound I-2.2HCl

FIG. 1 d: XRPD Compound I-2.HCl monohydrate

FIG. 2d : TGA Compound I-2.HCl monohydrate

FIG. 3d : DSC Compound I-2.HCl monohydrate

FIG. 1 e: XRPD Compound I-2.HCl.2H₂O

FIG. 2e : TGA Compound I-2.HCl.2H₂O

FIG. 3e : DSC Compound I-2.HCl.2H₂O

FIG. 4a : Solid State Compound I-1 free base

FIG. 4b : Solid State ¹³CNMR of Compound I-1.HCl

SUMMARY OF THE INVENTION

The present invention relates to processes and intermediates forpreparing compounds useful as inhibitors of ATR kinase, such asaminopyrazine-isoxazole derivatives and related moleculesAminopyrazine-isoxazole derivatives are useful as ATR inhibitors and arealso useful for preparing ATR inhibitors. The present invention alsorelates to solid forms of ATR inhibitors as well as deuterated ATRinhibitors.

One aspect of the invention provides a process for preparing a compoundof formula I:

comprising preparing a compound of formula 4:

from a compound of formula 3:

under suitable oxime formation conditions.

Another aspect comprises preparing a compound of formula 4:

from a compound of formula 3:

under suitable oxime formation conditions.

Another aspect of the present invention comprises a compound of formulaII:

or a pharmaceutically acceptable salt thereof, wherein each R^(1a),R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a),R^(9b), R¹⁰, R¹¹, R¹², and R¹³ is independently hydrogen or deuterium,and at least one of R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c),R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), R^(9b), R¹⁰, R¹¹. R¹², and R¹³ is deuterium.

Yet another aspect of the invention provides solid forms of a compoundof formula I-2:

Other aspects of the invention are set forth herein.

The present invention has several advantages over previously knownmethods. First, the present process has fewer number of total syntheticsteps compared with previously disclosed processes. Second, the presentprocess has improved yields over previously disclosed processes. Third,the present process is effective for compounds wherein R³ is a widerange of groups, such as alkyl groups or a large, hindered moiety, suchas a ring. Fourth, the present process comprises intermediates which aremore stable and have a longer shelf life. In certain embodiments, thenon-acidic formation of the oxime group in the present process allowsthe preservation of acid-sensitive protecting groups such as Boc or CBzduring the course of the synthesis. In other embodiments, the process ismore easily scaled up to larger quantities due to the elimination ofchromatography as a purification step.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention provides a process for making a compound ofpreparing a compound of formula 4:

from a compound of formula 3:

under suitable oxime formation conditions;

-   wherein    -   R¹ is C₁₋₆alkyl;    -   R² is C₁₋₆alkyl;    -   or R¹ and R², together with the oxygen atoms to which they are        attached, form an optionally substituted 5 or 6 membered        saturated heterocyclic ring having two oxygen atoms;    -   R³ is hydrogen, C₁₋₆alkyl, or a 3-6 membered saturated or        partially unsaturated heterocyclyl having 1-2 heteroatoms        selected from the group consisting of oxygen, nitrogen, and        sulfur; wherein the heterocyclyl is optionally substituted with        1 occurrence of halo or C₁₋₃alkyl;    -   J¹ is halo, C₁₋₄alkyl, or C₁₋₄alkoxy;    -   PG is a carbamate protecting group.

Another aspect provides a process for preparing a compound of formula I:

-   -   comprising the steps of:    -   preparing a compound of formula 4:

-   -   from a compound of formula 3:

-   -   under suitable oxime formation conditions;    -   wherein    -   R¹ is C₁₋₆alkyl;    -   R² is C₁₋₆alkyl;    -   or R¹ and R², together with the oxygen atoms to which they are        attached, form an optionally substituted 5 or 6 membered        saturated heterocyclic ring having two oxygen atoms;    -   R³ is hydrogen, C₁₋₆alkyl, or a 3-6 membered saturated or        partially unsaturated heterocyclyl having 1-2 heteroatoms        selected from the group consisting of oxygen, nitrogen, and        sulfur; wherein the heterocyclyl is optionally substituted with        1 occurrence of halo or C₁₋₃alkyl;    -   R⁴ is

-   -   Q is phenyl, pyridyl, or an N-alkylated pyridine;    -   J¹ is H, halo, C₁₋₄alkoxy, or C₁₋₄alkoxy;    -   J² is halo; CN; phenyl; oxazolyl; or a C₁₋₆aliphatic group        wherein up to 2 methylene units are optionally replaced with O,        NR″, C(O), S, S(O), or S(O)₂; said C₁₋₆aliphatic group is        optionally substituted with 1-3 fluoro or CN;    -   q is 0, 1, or 2;    -   PG is a carbamate protecting group.

Another embodiment further comprises the step of protecting a compoundof formula 2:

under suitable protection conditions to form the compound of formula 3.

Another embodiment further comprises the step of reacting a compound offormula 1:

with a suitable amine under suitable reductive amination conditions toform a compound of formula 2.

In some embodiments, the suitableamine is NHCH₃. In other embodiments,the suitable amine is

Another embodiment further comprises the step of reacting a compound offormula 4:

under suitable isoxazole formation conditions to form a compound offormula 5:

Another embodiment further comprises the step of reacting a compound offormula 5 under suitable coupling conditions followed by suitabledeprotection conditions to form a compound of formula I.

In some embodiments, PG is Boc or Cbz. In some embodiments, PG is Boc.

In other embodiments, R¹ is ethyl and R² ethyl.

In yet other embodiments, R³ is CH₃ or

In some embodiments, R⁴ is

wherein Q is phenyl. In some embodiments, Q is substituted in the paraposition with J₂, wherein q is 1.

In some embodiments, J¹ is H or halo. In some embodiments, J¹ is H. Inother embodiments, J¹ is halo.

In other embodiments, J² is a C₁₋₆aliphatic group wherein up to 1methylene unit is optionally replaced with S(O)₂. In some embodiments,J² is —S(O)₂—(C₁₋₅alkyl). In some embodiments, q is 1.

According to another embodiment,

-   -   R¹ is ethyl;    -   R² is ethyl;    -   R³ is CH₃ or

-   -   PG is Boc or Cbz;    -   J¹ is H;    -   R⁴ is

wherein Q is phenyl; J² is —S(O)₂—CH(CH₃)₂;

-   -   q is 1;

In some embodiments, R³ is CH₃. In some embodiments, R³ is CH₃. In yetanother embodiments, R³ is CH₃ or

According to another embodiment,

-   -   R¹ is ethyl;    -   R² is ethyl;    -   R³ is

-   -   PG is Boc;    -   J¹ is H.    -   R⁴ is

wherein Q is pyridyl; J² is

-   -   q is 1;

In some embodiments, R⁴ is

Reactions Conditions

In some embodiments, the suitable oxime formation conditions consist ofeither a single step sequence or a two step sequence.

In some embodiments, the two step sequence consists of firstdeprotecting the ketal group in the compound of formula 3 into analdehyde under suitable deprotection conditions, and then forming theoxime of formula 4 under suitable oxime formation conditions. In someembodiments, suitable deprotection conditions comprise adding catalyticamounts of para-toluenesulfonic acid (pTSA), acetone, and water; andsuitable oxime formation conditions comprise mixing togetherhydroxylamine, a catalytic amount of acid, a dehydrating agent, and analcoholic solvent. In other embodiments, the acid is pTSA or HCl, thedehydrating agent is molecular sieves or dimethoxyacetone, and thealcoholic solvent is methanol or ethanol.

In other embodiments, the single step sequence comprises addingNH₂OH.HCl and a mixture of THF and water. In other embodiments, thesequence comprises adding NH₂OH.HCl with a mixture of 2-methyltetrahydrofuran and water optionally buffered with Na₂SO₄. In someembodiments, 1 equivalent of the compound of formula 3 is combined witha 1.1 equivalents of NH₂OH.HCl in a 10:1 v/v mixture of THF and water.In some embodiments, 1 equivalent of the compound of formula 3 iscombined with a 1.1 equivalents of NH₂OH.HCl in a 10:1 v/v mixture of2-methyl tetrahydrofuran and water optionally buffered with Na₂SO₄.

In other embodiments, the protection conditions are selected from thegroup consisting of

-   -   R—OCOCl, a suitable tertiary amine base, and a suitable solvent;        wherein R is C₁₋₆alkyl optionally substituted with phenyl;    -   R(CO₂)OR′, a suitable solvent, and optionally a catalytic amount        of base, wherein R is and R′ are each independently C₁₋₆alkyl        optionally substituted with phenyl;    -   [RO(C═O)]₂O, a suitable base, and a suitable solvent.

In some embodiments, the suitable base is Et₃N, diisopropylamine, andpyridine; and the suitable solvent is selected from a chlorinatedsolvent, an ether, or an aromatic hydrocarbon. In other embodiments, thesuitable base is Et₃N, the suitable solvent is a chlorinated solventselected from DCM. In yet other embodiments, the protection conditionscomprise adding 1.20 equivalents of (Boc)₂O and 1.02 equivalents of Et₃Nin DCM.

According to another embodiment suitable coupling conditions compriseadding a suitable metal and a suitable base in a suitable solvent. Inother embodiments, the suitable metal is Pd[P(tBu)₃]₂; the suitablesolvent is a mixture of acetonitrile and water; and the suitable base issodium carbonate. In yet other embodiments, the suitable couplingconditions comprise adding 0.1 equivalents of Pd[P(tBu)₃]₂; 1 equivalentof boronic acid or ester; and 2 equivalents of sodium carbonate in a 2:1ratio v/v of acetonitrile/water at 60-70° C.

According to another embodiment, suitable deprotection conditionscomprise combining the compound of formula 5 with a suitable acid in asuitable solvent. In some embodiments, the suitable acid is selectedfrom para-toluenesulfonic acid (pTSA), HCl, TBAF, H₃PO₄, or TFA and thesuitable solvent is selected from acetone, methanol, ethanol, CH₂Cl₂,EtOAc, THF, 2-MeTHF, dioxane, toluene, or diethylether.

According to another embodiment, suitable isoxazole-formation conditionsconsists of two steps, the first step comprising reacting the compoundof formula 4 under suitable chlorooxime formation conditions to form achlorooxime intermediate; the second step comprising reacting thechlorooxime intermediate with acetylene under suitable cycloadditionconditions to form a compound of formula 5.

According to another embodiment, suitable chlorooxime formationconditions are selected from

-   -   N-chlorosuccinimide and suitable solvent or    -   potassium peroxymonosulfate, HCl, and dioxane.

In some embodiments, the suitable solvent is selected from a nonproticsolvent, an aromatic hydrocarbon, or an alkyl acetate. According toanother embodiment, the suitable chlorooxime formation conditions are1.05 equivalents of N-chlorosuccinimide in isopropylacetate at 40-50° C.

According to another embodiment, suitable cycloaddition conditionsconsist of a suitable base and a suitable solvent. In some embodiments,the suitable base is selected from pyridine, DIEA, TEA, t-BuONa, andK₂CO₃ and the suitable solvent is selected from acetonitrile,tetrahydrofuran, 2-methyltetrahydrofuran, MTBE, EtOAc, i-PrOAc, DCM,toluene, DMF, and methanol. In other embodiments, the suitable base isselected from Et₃N and the suitable solvent is selected from DCM.

According to another embodiment, the second step comprises reacting 1equivalent of acetylene with 1.2 equivalents of the chlorooximeintermediate and 1.3 equivalents of Et₃N in DCM at room temperature.

According to another embodiment, suitable isoxazole-formation conditionscomprise combining the compound of formula 4 with an oxidant in asuitable solvent. In som embodiments, said oxidant is[bis(trifluoroacetoxy)iodo] benzene and said solvent is a 1:1:1 mixtureof methanol, water, and dioxane.

Synthesis of Compounds I-2 and I-3

One embodiment provides a process for preparing a compound of formulaI-2:

comprising one or more of the following steps:

-   -   a) Reacting a compound of formula 1b:

with methylamine under suitable reductive amination conditions to form acompound of formula 2b:

-   -   b) reacting a compound of formula 2b under suitable Boc        protection conditions to form the compound of formula 3b:

-   -   c) reacting a compound of formula 3b under suitable oxime        formation conditions to form the compound of formula 4-i:

-   -   d) reacting a compound of formula 4-i under suitable chlorooxime        formation conditions to form the compound of formula 4-ii:

-   -   e) reacting the compound of formula 4-ii with a compound of        formula 4-iii

-   -   under suitable cycloaddition conditions to form a compound of        formula 4-iv:

-   -   f) .reacting a compound of formula 4-iv with a compound of        formula A-5-i:

-   -   under suitable coupling conditions to form the compound of        formula 5-i:

-   -   g) deprotecting a compound of formula 5-i under suitable Boc        deprotection conditions optionally followed by treatment under        basic aqueous conditions to form a compound of formula I-2.

Another embodiment provides a process for preparing a compound offormula I-3:

comprising one or more of the following steps:

-   -   a) Reacting a compound of formula A-1:

with tetrahydro-2H-pyran-4-amine under suitable reductive aminationconditions to form a compound of formula A-2:

-   -   b) reacting a compound of formula A-2 under suitable Boc        protection conditions to form the compound of formula A-3:

-   -   c) reacting a compound of formula A-3 under suitable oxime        formation conditions to form the compound of formula A-4:

-   -   d) reacting a compound of formula A-4:

-   -   under suitable chlorooxime formation conditions to form the        compound of formula A-4-i:

-   -   e) reacting the compound of formula A-4-i with a compound of        formula A-4-ii:

-   -   under suitable cycloaddition conditions to form the compound of        formula A-5:

-   -   f) reacting a compound of formula A-5 with a compound of formula        A-5-i:

-   -   under suitable coupling conditions to form the compound of        formula A-6:

-   -   g) deprotecting a compound of formula A-6 under suitable Boc        deprotection conditions optionally followed by treatment under        basic aqueous conditions to form a compound of formula I-3.

Suitable coupling conditions comprise combining a suitable palladiumcatalyst with a suitable base in a suitable solvent. Suitable palladiumcatalyst include, but are not limited to, Pd[P(tBu)₃]₂, Pd(dtbpf)Cl₂,Pd(PPh₃)₂Cl₂, Pd(PCy₃)₂Cl₂ , Pd(dppf)Cl₂, and Pd(dppe)Cl₂. Suitablesolvents include, but are not limited to. toluene, MeCN, water, EtOH,IPA, 2-Me-THF, or IPAc. Suitable bases include, but are not limited to,K₂CO₃, Na₂CO₃, or K₃PO₄.

Suitable oxime formation conditions consist of either a single stepsequence or a two step sequence. The two step sequence consists of firstdeprotecting the ketal group in the compound of formula A-3 into analdehyde under suitable deprotection conditions, and then forming theoxime of formula A-4 under suitable oxime formation conditions.

The single step sequence comprises, for example, comprise mixingtogether hydroxylamine, an acid, an organic solvent, and water. In someembodiments, NH₂OH.HCl is added to a mixture of THF and water. In someembodiments, 1 equivalent of the compound of formula 3-A is combinedwith a 1.1 equivalents of NH₂OH.HCl in a 10:1 v/v mixture of THF/water.

Suitable deprotection conditions comprise adding an acid, acetone, andwater. Suitable acids include pTSA or HCl, tsuitable organic solventsinclude chlorinated solvents (e.g., dichloromethane (DCM),dichloroethane (DCE), CH₂Cl₂, and chloroform); an ether (e.g., THF,2-MeTHF and dioxane); an aromatic hydrocarbons (e.g., toluene andxylenes, or other aprotic solvents.

Suitable cycloaddition conditions comprise a suitable base (e.g.,pyridine, DIEA, TEA, t-BuONa, or K₂CO₃) and a suitable solvent (e.g.,acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, MTBE, EtOAc,i-PrOAc, DCM, toluene, DMF, and methanol_.

Suitable chlorooxime formation conditions comprise adding HCl in dioxameto a solution of the oxime in the presence of NCS in a suitable solventselected from a nonprotic solvents (DCM, DCE, THF, and dioxane),aromatic hydrocarbons (e.g. toluene, xylenes), and alkyl acetates (e.g.,isopropyl acetate, ethyl acetate).

Suitable Boc deprotection conditions comprises adding a suitable Bocdeprotecting agent (e.g, TMS-Cl, HCl, TBAF, H₃PO₄, or TFA) and asuitable solvent (e.g., acetone, toluene, methanol, ethanol, 1-propanol,isopropanol, CH₂Cl₂, EtOAc, isopropyl acetate, tetrahydrofuran,2-methyltetraydrofuran, dioxane, and diethylether). In some embodiments,the suitable Boc deprotection conditions comprises adding a suitable Bocdeprotecting agent selected from HCl, TFA and a suitable solventselected from acetone, toluene, isopropyl acetate, tetrahydrofuran, or2-methyltetraydrofuran.

Suitable Boc protection conditions include (Boc)₂O, a suitable base, anda suitable solvent. Suitable bases include, but are not limited to,Et₃N, diisopropylamine, and pyridine. Suitable solvents include, but arenot limited to, chlorinated solvents (e.g., dichloromethane (DCM),dichloroethane (DCE), CH₂Cl₂, and chloroform); an ether (e.g., THF,2-MeTHF and dioxane); an aromatic hydrocarbons (e.g., toluene andxylenes, or other aprotic solvents. In some embodiments, the suitablebase is Et₃N, the suitable solvent is DCM, tetrahydrofuran or2-methyltetrahydrofuran. In certain embodiments, the protectionconditions comprise adding 1.05 equivalents of (Boc)₂O in2-methyltetrahydrofuran or DCM.

Suitable reductive amination conditions comprise adding a reducing agentselected from NaBH₄ NaBH₄, NaBH₃CN, or NaBH(OAc)₃ in the presence of asolvent selected from dichloromethane (DCM), dichloroethane (DCE), analcoholic solvent selected from methanol, ethanol, 1-propanol,isopropanol, or a nonprotic solvent selected from dioxane,tetrahydrofuran, or 2-methyltetrahydrofuran and optionally a baseselected from Et₃N or diisopropylethylamine. In some embodiments, thesuitable reductive amination conditions comprise adding 1.2 equivalentsof NaBH₄ caplets in the presence Et₃N in MeOH.

Another aspect of the present invention provides a compound of FormulaII:

or a pharmaceutically acceptable salt thereof,

wherein each R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵,R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) is independentlyhydrogen or deuterium, and

at least one of R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁵,R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) is deuterium.

In some embodiments, R⁹a and R^(9b) are the same. In other embodiments,R^(9a) and R^(9b) are deuterium and R^(1a), R^(1b), R^(1c), R², R^(3a),R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁰, R^(11a), R^(11b), R^(12a),R^(12b), R^(13a), R^(13b), R^(14a), and R^(14b) are deuterium orhydrogen. In yet another embodiment, R^(9a) and R^(9b) are deuterium andR^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸,R¹⁰, R^(11a), R^(11b), R^(12a), R^(12b), R^(13a), R^(13b), R^(14a), andR^(14b) are hydrogen.

In one embodiment, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are thesame. In another embodiment R^(9a), R^(9b), R^(10a), R^(10b), andR^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b),R^(3c), R⁴, R⁵, R⁶, R⁷, and R⁸ are deuterium or hydrogen. In someembodiments, R^(9a), R^(9b), R^(10a), R^(10b), and R^(110c) aredeuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴,R⁵, R⁶, R⁷, and R⁸ are hydrogen.

In other embodiments, R^(10a), R^(10b), and R^(10c) are the same. In oneembodiment, R^(10a), R^(10b), and R^(10c) are deuterium, and R^(1a),R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a),and R^(9b) are deuterium or hydrogen. In yet another embodiment,R^(10a), R^(10b), and R^(10c) are deuterium, and R^(1a), R^(1b), R^(1c),R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a) and R^(9b) arehydrogen.

In some embodiments, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), andR^(3c) are the same. In another embodiment R^(1a), R^(1b), R^(1c), R²,R^(3a), R^(3b), and R^(3c) are deuterium R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a),R^(9b), R^(10a), R^(10b), and R^(10c) are deuterium or hydrogen. In yetanother embodiment, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R⁴, R⁵,R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are deuterium.

In another embodiment, R⁶ is deuterium, and R^(1a), R^(1b), R^(1c), R²,R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁷, R⁸, R^(9a), R^(9b), R^(10a),R^(10b), and R^(10c) are deuterium or hydrogen. In yet anotherembodiment, R⁶ is deuterium, and R_(1a), R^(1b), R², R^(3a), R^(3c), R⁴,R⁵, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are hydrogen.

In other embodiments, R² is deuterium, and R^(1a), R^(1b), R^(1c),R^(3a), R^(3b), and R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a),R^(10b), and R^(10c) are deuterium or hydrogen. In another embodiment,R² is deuterium, and R^(1a), R^(1b), R^(1c), R^(3a), R^(3b), and R^(3c),R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) arehydrogen.

In another embodiment, R⁷ is deuterium, and R^(1a), R^(1b), R^(1c), R²,R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁸, R^(9a), R^(9b), R^(10a),R^(10b), and R^(10c) are deuterium or hydrogrn. In other embodiments, R⁷is deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c),R⁴, R⁵, R⁶, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), R^(10c), are hydrogen.

In yet another embodiment, R⁸ is deuterium, and R^(1a), R^(1b), R^(1c),R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R^(9a), R^(9b), R^(10a),R^(10b), and R^(10c) are deuterium or hydrogen. In another embodiment,R⁸ is deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c),R⁴, R⁵, R⁶, R⁷, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) arehydrogen.

In some embodiments, at least one of R^(10a), R^(10b), or R^(10c) arethe same. In another embodiment, at least one of R^(10a), R^(10b), orR^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b),R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), and R^(9b), are deuterium orhydrogen. In yet another embodiment, at least one of R^(10a), R^(10b),or R^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a),R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), and R^(9b) are hydrogen.

In some embodiments, at least two of R^(10a), R^(10b), or R^(10c) arethe same. In another embodiment, at least two of R^(10a), R^(10b), orR^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b),R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), and R^(9b), are deuterium orhydrogen. In yet another embodiment, at least one of R^(10a), R^(10b),or R^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a),R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), and R^(9b) are hydrogen.

In another embodiment, R^(1a), R^(1b), R^(1c), R^(3a), R^(3b), andR^(3c) are the same. In some embodiments, R^(1a), R^(1b), R^(1c),R^(3a), R^(3b), and R^(3c) are deuterium, and R², R⁴, R⁵, R⁶, R⁷, R⁸,R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are deuterium or hydrogen.In yet another embodiment, R^(1a), R^(1b), R^(1c), R^(3a), R^(3b), andR^(3c) are deuterium, and R², R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), R^(10a),R^(10b), and R^(10c) are hydrogen.

In yet another embodiment, R⁴ is deuterium, and R^(1a), R^(1b), R^(1c),R², R^(3a), R^(3b), R^(3c), R⁵, R⁶, R⁷, R⁸, R^(9a), R^(10a), R^(10b),and R^(10c) are deuterium or hydrogen. In other embodiments, R⁴ isdeuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁵,R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are hydrogen.

In another embodiment, R⁵ is deuterium, and R^(1a), R^(1b), R^(1c), R²,R^(3a), R^(3b), R^(3c), R⁴, R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a),R^(10b), and R^(10c) are deuterium or hydrogen. In yet anotherembodiment, R⁵ is deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a),R^(3b), R^(3c), R⁴, R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), andR^(10c) are hydrogen.

In another embodiment, at least one of R^(9a) or R^(9b) are the same. Inother embodiments, at least one of R^(9a) or R^(9b) are deuterium, andR^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸,R^(10a), R^(10b), and R^(10c) are deuterium or hydrogen. In someembodiments, at least one of R^(9a) and R^(9b) are deuterium, andR^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸,R^(10a), R^(10b), and R^(10c) are hydrogen.

In one embodiment, R⁶, R^(9a) and R^(9b) are the same. In someembodiments, R⁶, R^(9a) and R^(9b) are deuterium, and R^(1a), R^(1b),R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁷, R⁸, R^(10a), R^(10b),and R^(10c) are deuterium or hydrogen. In other embodiments, R⁶, R^(9a)and R^(9b) are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a),R^(3b), R^(3c), R⁴, R⁵, R⁷, R⁸, R^(10a), R^(10b), and R^(10c) arehydrogen.

In some embodiments, R², R^(10a), R^(10b), and R^(10c) are the same. Inanother embodiment, R², R^(10a), R^(10b), and R^(10c) are deuterium, andR^(1a), R^(1b), R^(1c), R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸,R^(9a), and R^(9b) are deuterium or hydrogen. In yet another embodiment,R², R^(10a), R^(10b), R^(10c) are deuterium, and R^(1a), R^(1b), R^(1c),R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), and R^(9b) arehydrogen.

In some embodiments, R⁷ and at least two of R^(10a), R^(10b), or R^(10c)are the same. In another embodiment, R⁷ and at least two of R^(10a),R^(10b), or R^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R²,R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁸, R^(9a), and R^(9b) are deuteriumor hydrogen. In yet another embodiment, R⁷ and at least two of R^(10a),R^(10b), or R^(10c) are deuterium, and R^(1a), R^(1b), R^(1c), R²,R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁸, R^(9a), and R^(9b) are hydrogen.

In some embodiments, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b)R^(3c),and at least one of R^(10a), R^(10b), or R^(10c) are the same. Inanother embodiment, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b)R^(3c),and at least one of R^(10a), R^(10b), R^(10c) are deuterium, and R⁴, R⁵,R⁶, R⁷, R⁸, R^(9a), and R^(9b) are deuterium or hydrogen. In yet anotherembodiment, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b)R^(3c), and atleast one of R^(10a), R^(10b), or R^(10c) are deuterium, and R⁴, R⁵, R⁶,R⁷, R⁸, R^(9a), and R^(9b) are hydrogen.

In some embodiments, R^(1a), R^(1b), R^(1c), R^(3a), R^(3b)R^(3c), andR⁵ are the same. In another embodiment, R^(1a), R^(1b), R^(1c), R^(3a),R^(3b)R^(3c), and R⁵ are deuterium, and R², R⁴, R⁶, R⁷, R⁸, R^(9a),R^(9b), R^(10a), R^(10b), and R^(10c) are deuterium or hydrogen. In yetanother embodiment, R^(1a), R^(1b), R^(1c), R^(3a), R^(3b), R^(3c), andR⁵ are deuterium, and R², R⁴, R⁶, R⁷, R⁸, R^(9a), R^(9b), R^(10a),R^(10b), and R^(10c) are hydrogen.

In other embodiments, R⁴ and R⁶ are the same. In another embodiment, R⁴and R⁶ are deuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b),R^(3c), R⁵, R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) aredeuterium or hydrogen. In yet another embodiment, R⁴ and R⁶ aredeuterium, and R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁵,R⁷, R⁸, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are hydrogen.

In one embodiment, R², R⁵, R^(9a), and R^(9b) are the same. In someembodiments, R², R⁵, R^(9a), and R^(9b) are deuterium, and R^(1a),R^(1b), R^(1c), R^(3a), R^(3b), R^(3c), R⁴, R⁶, R⁷, R⁸, R^(10a),R^(10b), and R^(10c) are deuterium or hydrogen. In another embodiment,R², R⁵, R^(9a), and R^(9b) are deuterium, and R^(1a), R^(1b), R^(1c),R^(3a), R^(3b), R^(3c), R⁴, R⁶, R⁷, R⁸, R^(10a), R^(10b), and R^(10c)are hydrogen.

In yet another embodiment, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b),R^(3c), R⁵, R⁶, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) are thesame. In some embodiments, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b),R^(3c), R⁵, R⁶, R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) aredeuterium, and R⁴, R⁷, and R⁸ are deuterium or hydrogen. In otherembodiments, R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁵, R⁶,R^(9a), R^(9b), R^(10a), R^(10b), and R^(10c) is deuterium, and R⁴, R⁷,and R⁸ are hydrogen.

In some embodiments, the variables are as depicted in the compounds ofthe disclosure including compounds in the tables below.

TABLE I

I-1

I-2

I-3

TABLE II

II-1

II-2

II-3

II-4

II-5

II-6

II-7

II-8

II-9

II-10

II-11

II-12

II-13

II-14

II-15

II-16

II-17

II-18

II-19

II-20

II-21

II-22

Compounds of this invention include those described generally herein,and are further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As described herein, a specified number range of atoms includes anyinteger therein. For example, a group having from 1-4 atoms could have1, 2, 3, or 4 atoms.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally herein, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds.

Unless otherwise indicated, a substituent connected by a bond drawn fromthe center of a ring means that the substituent can be bonded to anyposition in the ring. In example i below, for instance, J¹ can be bondedto any position on the pyridyl ring. For bicyclic rings, a bond drawnthrough both rings indicates that the substituent can be bonded from anyposition of the bicyclic ring. In example ii below, for instance, J¹ canbe bonded to the 5-membered ring (on the nitrogen atom, for instance),and to the 6-membered ring.

The term “stable”, as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, recovery, purification, and use for one or moreof the purposes disclosed herein. In some embodiments, a stable compoundor chemically feasible compound is one that is not substantially alteredwhen kept at a temperature of 40° C. or less, in the absence of moistureor other chemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched), branched, or cyclic, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation that has a single point ofattachment to the rest of the molecule.

Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. Aliphatic groupsmay be linear or branched, substituted or unsubstituted alkyl, alkenyl,or alkynyl groups. Specific examples include, but are not limited to,methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl,ethynyl, and tert-butyl. Aliphatic groups may also be cyclic, or have acombination of linear or branched and cyclic groups. Examples of suchtypes of aliphatic groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, —CH₂—cyclopropyl,CH₂CH₂CH(CH₃)-cyclohexyl.

The term “cycloaliphatic” (or “carbocycle” or “carbocyclyl”) refers to amonocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Examples of cycloaliphatic groups include, butare not limited to, cycloalkyl and cycloalkenyl groups. Specificexamples include, but are not limited to, cyclohexyl, cyclopropenyl, andcyclobutyl.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinmeans non-aromatic, monocyclic, bicyclic, or tricyclic ring systems inwhich one or more ring members are an independently selected heteroatom.In some embodiments, the “heterocycle”, “heterocyclyl”, or“heterocyclic” group has three to fourteen ring members in which one ormore ring members is a heteroatom independently selected from oxygen,sulfur, nitrogen, or phosphorus, and each ring in the system contains 3to 7 ring members.

Examples of heterocycles include, but are not limited to,3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl,2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl,2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl,4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl,4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,5-imidazolidinyl, indolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and1,3-dihydro-imidazol-2-one.

Cyclic groups, (e.g. cycloaliphatic and heterocycles), can be linearlyfused, bridged, or spirocyclic.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation. As would be known by one of skill in theart, unsaturated groups can be partially unsaturated or fullyunsaturated. Examples of partially unsaturated groups include, but arenot limited to, butene, cyclohexene, and tetrahydropyridine. Fullyunsaturated groups can be aromatic, anti-aromatic, or non-aromatic.Examples of fully unsaturated groups include, but are not limited to,phenyl, cyclooctatetraene, pyridyl, thienyl, and1-methylpyridin-2(1H)-one.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached through an oxygen (“alkoxy”) orsulfur (“thioalkyl”) atom.

The terms “haloalkyl”, “haloalkenyl”, “haloaliphatic”, and “haloalkoxy”mean alkyl, alkenyl or alkoxy, as the case may be, substituted with oneor more halogen atoms. This term includes perfluorinated alkyl groups,such as —CF₃ and —CF₂CF₃.

The terms “halogen”, “halo”, and “hal” mean F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”. Examples of heteroaryl rings include, butare not limited to, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl),2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl),triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl,benzofuryl, benzothiophenyl, indolyl (e.g., 2-indolyl), pyrazolyl (e.g.,2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl,1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl,4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,3-isoquinolinyl, or 4-isoquinolinyl).

It shall be understood that the term “heteroaryl” includes certain typesof heteroaryl rings that exist in equilibrium between two differentforms. More specifically, for example, species such hydropyridine andpyridinone (and likewise hydroxypyrimidine and pyrimidinone) are meantto be encompassed within the definition of “heteroaryl.”

The term “protecting group” and “protective group” as used herein, areinterchangeable and refer to an agent used to temporarily block one ormore desired functional groups in a compound with multiple reactivesites. In certain embodiments, a protecting group has one or more, orpreferably all, of the following characteristics: a) is addedselectively to a functional group in good yield to give a protectedsubstrate that is b) stable to reactions occurring at one or more of theother reactive sites; and c) is selectively removable in good yield byreagents that do not attack the regenerated, deprotected functionalgroup. As would be understood by one skilled in the art, in some cases,the reagents do not attack other reactive groups in the compound. Inother cases, the reagents may also react with other reactive groups inthe compound. Examples of protecting groups are detailed in Greene, T.W., Wuts, P. G in “Protective Groups in Organic Synthesis”, ThirdEdition, John Wiley & Sons, New York: 1999 (and other editions of thebook), the entire contents of which are hereby incorporated byreference. The term “nitrogen protecting group”, as used herein, refersto an agent used to temporarily block one or more desired nitrogenreactive sites in a multifunctional compound. Preferred nitrogenprotecting groups also possess the characteristics exemplified for aprotecting group above, and certain exemplary nitrogen protecting groupsare also detailed in Chapter 7 in Greene, T. W., Wuts, P. G in“Protective Groups in Organic Synthesis”, Third Edition, John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

In some embodiments, a methylene unit of an alkyl or aliphatic chain isoptionally replaced with another atom or group. Examples of such atomsor groups include, but are not limited to, nitrogen, oxygen, sulfur,—C(O)—, —C(═N—CN)—, —C(═NR)—, —C(═NOR)—, —SO—, and —SO₂—. These atoms orgroups can be combined to form larger groups. Examples of such largergroups include, but are not limited to, —OC(O)—, —C(O)CO—, —CO₂—,—C(O)NR—, —C(═N—CN), —NRCO—, —NRC(O)O—, —SO₂NR—, —NRSO₂—, —NRC(O)NR—,—OC(O)NR—, and —NRSO₂NR—, wherein R is, for example, H or C₁₋₆aliphatic.It should be understood that these groups can be bonded to the methyleneunits of the aliphatic chain via single, double, or triple bonds. Anexample of an optional replacement (nitrogen atom in this case) that isbonded to the aliphatic chain via a double bond would be —CH₂CH═N—CH₃.In some cases, especially on the terminal end, an optional replacementcan be bonded to the aliphatic group via a triple bond. One example ofthis would be CH₂CH₂CH₂C≡N. It should be understood that in thissituation, the terminal nitrogen is not bonded to another atom.

It should also be understood that, the term “methylene unit” can alsorefer to branched or substituted methylene units. For example, in anisopropyl moiety [—CH(CH₃)₂], a nitrogen atom (e.g. NR) replacing thefirst recited “methylene unit” would result in dimethylamine [—N(CH₃)₂].In instances such as these, one of skill in the art would understandthat the nitrogen atom will not have any additional atoms bonded to it,and the “R” from “NR” would be absent in this case.

Unless otherwise indicated, the optional replacements form a chemicallystable compound. Optional replacements can occur both within the chainand/or at either end of the chain; i.e. both at the point of attachmentand/or also at the terminal end. Two optional replacements can also beadjacent to each other within a chain so long as it results in achemically stable compound. For example, a C₃ aliphatic can beoptionally replaced by 2 nitrogen atoms to form —C—N≡N. The optionalreplacements can also completely replace all of the carbon atoms in achain. For example, a C₃ aliphatic can be optionally replaced by —NR—,—C(O)—, and —NR— to form —NRC(O)NR— (a urea).

Unless otherwise indicated, if the replacement occurs at the terminalend, the replacement atom is bound to a hydrogen atom on the terminalend. For example, if a methylene unit of —CH₂CH₂CH₃ were optionallyreplaced with —O—, the resulting compound could be —OCH₂CH₃, —CH₂OCH₃,or —CH₂CH₂OH. It should be understood that if the terminal atom does notcontain any free valence electrons, then a hydrogen atom is not requiredat the terminal end (e.g., —CH₂CH₂CH═O or —CH₂CH₂C≡N).

Unless otherwise indicated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, geometric,conformational, and rotational) forms of the structure. For example, theR and S configurations for each asymmetric center, (Z) and (E) doublebond isomers, and (Z) and (E) conformational isomers are included inthis invention. As would be understood to one skilled in the art, asubstituent can freely rotate around any rotatable bonds. For example, asubstituent drawn as

also represents

Therefore, single stereochemical isomers as well as enantiomeric,diastereomeric, geometric, conformational, and rotational mixtures ofthe present compounds are within the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

In the compounds of this invention any atom not specifically designatedas a particular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Also unlessotherwise stated, when a position is designated specifically as “D” or“deuterium”, the position is understood to have deuterium at anabundance that is at least 3340 times greater than the natural abundanceof deuterium, which is 0.015% (i.e., at least 50.1% incorporation ofdeuterium).

“D” and “d” both refer to deuterium.

Additionally, unless otherwise indicated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

Processes

Processes and compounds described herein are useful for producing ATRinhibitors that contain an aminopyrazine-isoxazole core. The generalsynthetic procedures shown in schemes herein are useful for generating awide array of chemical species which can be used in the manufacture ofpharmaceutical compounds.

Step 1

The compound of formula I can be made according to the steps outlined inScheme A. Step 1 depicts the use of a readily available aldehyde/ketalas a starting point for the preparation of compounds of formula I, I-A,and I-B. Reductive amination between compound 1 and a suitable primaryamine, under conditions known to those skilled in the art leads tocompound 2 where a benzylamine motif has been installed. For example,imines can be formed by combining an amine and an aldehyde in a suitablesolvent, such as dichloromethane (DCM), dichloroethane (DCE), analcoholic solvent (e.g., methanol, ethanol), or a nonprotic solvent(e.g., dioxane or tetrahydrofuran (THF)). These imines can then bereduced by known reducing agents including, but not limited to, NaBH₄,NaBH₃CN, and NaBH(OAc)₃ (see JOC 1996, 3849). In some embodiments, 1.05equivalents of amine is combined with 1 equivalent of aldehyde inmethanol. In other embodiments, 1.2 equivalents of amine is combinedwith 1 equivalent of aldehyde in methanol. This step is then followed byreduction with 0.6 to 1.4 (such as 1.2) equivalents of NaBH₄. In somecases, if an amine salt is used, base (e.g., Et₃N ordiisopropylethylamine) can also be added.

Step 2

Step 2 depicts the protection of the benzylamine 1 prepared above, usinga carbamate-based protecting group, under suitable protection conditionsknown to those skilled in the art. Various protecting groups, such asCbz and Boc, can be used. Protection conditions include, but are notlimited to the following:

-   -   a) R—OCOCl, a suitable tertiary amine base, and a suitable        solvent; wherein R is C₁₋₆alkyl optionally substituted with        phenyl;    -   b) R(CO₂)OR′, a suitable solvent, and optionally a catalytic        amount of base, wherein R is and R′ are each independently        C₁₋₆alkyl optionally substituted with phenyl;    -   c) [RO(C═O)]₂O, a suitable base, and a suitable solvent.

Examples of suitable bases include, but are not limited to, Et₃N,diisopropylamine, and pyridine. Examples of suitable solvents includechlorinated solvents (e.g., dichloromethane (DCM), dichloroethane (DCE),CH₂Cl₂, and chloroform), ethers (e.g., THF, 2-MeTHF, and dioxane),aromatic hydrocarbons (e.g., toluene, xylenes) and other aproticsolvents.

In some embodiments, protection can be done by reacting the benzylaminewith (Boc)₂O and Et₃N in DCM. In some embodiments, 1.02 equivalents of(Boc)₂O and 1.02 equivalents of Et₃N 1.02 are used. In anotherembodiment, protection can be done by reacting the benzylamine with(Boc)₂O in 2-MeTHF. In some embodiments, 1.05 equivalents of (Boc)₂O areused.

Step 3

Step 3 shows how the ketal functional group in 3 is then converted intothe oxime 4 in a single step. This direct conversion from ketal to oximeis not extensively described in the literature and it will beappreciated that this step could also be conducted in a two-stepsequence, transiting through the aldehyde after deprotection of theketal using methodologies known to those skilled in the art.

Oxime formation conditions comprise mixing together hydroxylamine, acid,optionally a dehydrating agent, and an alcoholic solvent. In someembodiments, the acid is a catalytic amount. In some embodiments, theacid is pTSA or HCl, the dehydrating agent is molecular sieves ordimethoxyacetone, and the alcoholic solvent is methanol or ethanol. Insome embodiments, the hydroxylamine hydrochloride is used in which caseno additional acid is required. In other embodiments, the desiredproduct is isolated via a biphasic work up and optionally precipitationor crystallization. If a biphasic work up is used, a dehydrating agentis not needed.

In another embodiment, the oxime formation conditions comprise of mixingtogether hydroxylamine, an acid, an organic solvent and water. Examplesof suitable organic solvents include chlorinated solvents (e.g.,dichloromethane (DCM), dichloroethane (DCE), CH₂Cl₂, and chloroform),ethers (e.g., THF, 2-MeTHF and dioxane), aromatic hydrocarbons (e.g.,toluene, xylenes) and other aprotic solvents. In some embodiments, 1.5equivalents of hydroxylamine hydrochloride are used, the organic solventis 2-MeTHF and the water is buffered with Na₂SO₄. In another embodiment,1.2 equivalents of hydroxylamine hydrochloride are used, the organicsolvent is THF.

In some embodiments, suitable deprotection conditions comprise addingcatalytic amounts of para-toluenesulfonic acid (pTSA), acetone, andwater; and then forming the oxime using conditions known to one skilledin the art. In other embodiments, a single step sequence is used. Insome embodiments, the single step sequence comprises adding NH₂OH.HCland a mixture of THF and water. In some embodiments, 1 equivalent of thecompound of formula 3 is combined with a 1.1 equivalents of NH₂OH.HCl ina 10:1 v/v mixture of THF/water.

Step 4

Step 4 illustrates how the oxime 4 is then transformed and engaged in a[3+2] cycloaddition to for the isoxazole 5. This transformation can beconducted in one pot but requires two distinct steps. The first step isan oxidation of the oxime functional group into a nitrone, or a similarintermediate with the same degree of oxidation, for example achlorooxime. This reactive species then reacts with an alkyne in a [3+2]cycloaddition to form the isoxazole adduct.

In some embodiments, the suitable isoxazole-formation conditionsconsists of two steps, the first step comprising reacting the compoundof formula 4 under suitable chlorooxime formation conditions to form achlorooxime intermediate; the second step comprising reacting thechlorooxime intermediate with acetylene under suitable cycloadditionconditions to form a compound of formula 5.

In some embodiments, the chlorooxime formation conditions are selectedfrom

a) N-chlorosuccinimide and suitable solvent;

b) potassium peroxymonosulfate, HCl, and dioxane; and

c) Sodium hypochlorite and a suitable solvent

Examples of suitable solvents include, but are not limited to, nonproticsolvents (e.g., DCM, DCE, THF, 2-MeTHF, MTBE and dioxane), aromatichydrocarbons (e.g. toluene, xylenes), and alkyl acetates (e.g.,isopropyl acetate, ethyl acetate).

Isolation of the product can be achieved by adding an antisolvent to asolution of a compound of formula 5. Examples of suitable solvents forisolating the chlorooxime intermediate include mixtures of suitablesolvents (EtOAc, IPAC) with hydrocarbons (e.g., hexanes, heptane,cyclohexane), or aromatic hydrocarbons (e.g., toluene, xylenes). In someembodiments, heptane is added to a solution of chlorooxime in IPAC.

Suitable cycloaddition conditions consist of combining the chlorooximewith acetylene with a suitable base and a suitable solvent. Suitablesolvents include protic solvents, aproptic solvents, polar solvents, andnonpolar solvents. Examples of suitable solvent include, but are notlimited to, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran,MTBE, EtOAc, i-PrOAc, DCM, toluene, DMF, and methanol. Suitable basesinclude, but are not limited to, pyridine, DIEA, TEA, t-BuONa, andK₂CO₃. In some embodiments, suitable cycloaddition conditions compriseadding 1.0 equivalents of chlorooxime, 1.0 equivalents of acetylene, 1.1equivalents of Et3N in DCM.

Isolation of the product can be achieved by adding an antisolvent to asolution of a compound of formula 5. Examples of suitable solvents forisolating the chlorooxime include mixtures of suitable solvents (EtOAc,IPAC) with hydrocarbons (e.g., hexanes, heptane, cyclohexane), oraromatic hydrocarbons (e.g., toluene, xylenes). In some embodiments,heptane is added to a solution of chlorooxime in IPAC.

Step 5

Step 5 depicts the final step(s) of the preparation of compounds offormula I. When the R4 group is bromo, intermediate 5 can be subjectedto a Suzuki cross-coupling with boronic acid or esters, under conditionsknown to those skilled in the art, to form compounds where R4 an aryl,heteroaryl or alternative moieties resulting from the metal-assistedcoupling reaction. When intermediate 5 is suitably functionalised, adeprotection step can be carried out to remove the protecting groups andgenerate the compounds of formula I.

Metal assisted coupling reactions are known in the art (see e.g., Org.Proc. Res. Dev. 2010, 30-47). In some embodiments, suitable couplingconditions comprise adding 0.1 equivalents of Pd[P(tBu)₃]₂; 1 equivalentof boronic acid or ester; and 2 equivalents of sodium carbonate in a 2:1ratio v/v of acetonitrile/water at 60-70° C. In other embodiments,suitable coupling conditions comprise adding 0.010-0.005 equivalentsPd(dtbpf)Cl₂, 1 equivalent of boronic acid or ester, and 2 equivalentsof potassium carbonate in a 7:2 v/v of toluene and water at 70° C.

The final product can treated with a metal scavenger (silica gel,functionalized resins, charcoal) (see e.g., Org. Proc. Res. Dev. 2005,198-205). In some embodiments, the solution of the product is treatedwith Biotage MP-TMT resin.

The product can also be isolated by crystallization from an alcoholicsolvent (e.g. methanol, ethanol, isopropanol). In some embodiments thesolvent is ethanol. In other embodiments the solvent is isopropanol.

Deprotection of Boc groups is known in the art (see e.g. ProtectingGroups in Organic Synthesis, Greene and Wuts). In some embodiments,suitable deprotection conditions are hydrochloric acid in acetone at35-45° C. In other embodiments, suitable deprotection conditions are TFAin DCM.

Step 6

Step 6 illustrates how compounds of formula I are converted to compoundsof formula I-A using a base under suitable conditions known to thoseskilled in the art. In some embodiments, isolation of the free-base formof compounds of formula I may be achieved by adding suitable base, suchas NaOH to an alcoholic acidic solution of compounds of formula I toprecipitate the product.

Step 7

Step 7 illustrates how compounds of formula I-A are converted tocompounds of formula I-B using an acid under syuitable conditions knownto those skilled in the art.

In some embodiments suitable conditions involve adding aqueous HCl, to asuspension of compounds of formula I-A in acetone at 35° C. then heatingat 50° C.

Scheme B shows a general synthetic method for the preparation ofd1-boronate intermediates. A suitable 1-halo-(isopropylsulfonyl)benzeneis treated with a base such as, but not limited to NaH, LiHMDS or KHMDSfollowed by quenching of the anion with deuterium source such as D₂O.The halogen is then transformed into a suitable boronate derivative via,for example, metal mediated cross-coupling catalyzed by, for instance,Pd(^(t)Bu₃)₂ or Pd(dppf)Cl₂.DCM.

Scheme C shows a general synthetic method for the preparation ofd6-boronate intermediates. A suitable 1-halo-(methylsulfonyl)benzene istreated with a base such as, but not limited to NaH, LiHMDS or KHMDSfollowed by quenching of the anion with deuterium source such as D₃CI.This reaction is repeated until the desired amount of deuterium has beenincorporated into the molecule. The halogen is then transformed into asuitable boronate derivative via, for example, metal mediatedcross-coupling catalyzed by, for instance, Pd(^(t)Bu₃)₂ orPd(dppf)Cl₂.DCM.

Scheme D shows a general synthetic method for the preparation ofd7-boronate intermediates. 4-Bromobenzenethiol is treated with a basesuch as, but not limited to NaH, LiHMDS or KHMDS followed by quenchingof the anion with deuterium source such as1,1,1,2,3,3,3-heptadeuterio-2-iodo-propane. The sulfide is then oxidizedto the corresponding sulfone using, for example, mCPBA or Oxone. Thehalogen is then transformed into a suitable boronate derivative via, forexample, metal mediated cross-coupling catalyzed by, for instance,Pd(^(t)Bu₃)₂ or Pd(dppf)Cl₂.DCM.

Scheme E shows a general synthetic method for the preparation ofboronate intermediates where the aryl ring is substituted with adeuterium. A suitable 1-iodo-4-bromo-aryl derivative is treated with asubstituted thiol such as propane-2-thiol under metal catalyzed couplingconditions using a catalyst such as CuI. The sulfide is then oxidized tothe corresponding sulfone using, for example, mCPBA or Oxone. Thebromide is then transformed into a suitable boronate derivative via, forexample, metal mediated cross-coupling catalyzed by, for instance,Pd(^(t)Bu₃)₂ or Pd(dppf)Cl₂.DCM. The remaining substituent is thenconverted into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitbale metal catalyst, such as Pdon C under an atmposhere of deuterium gas. In addition, the1-bromo-(isopropylsulfonyl)benzene can be treated with a base such as,but not limited to NaH, LiHMDS or KHMDS followed by quenching of theanion with deuterium source such as D₂O. The bromide is then transformedinto a suitable boronate derivative via, for example, metal mediatedcross-coupling catalyzed by, for instance, Pd(^(t)Bu₃)₂ orPd(dppf)Cl₂.DCM. The remaining substituent is then converted intodeuterium by, for instance, metal catalyzed halogen-deuterium exchangeusing a suitbale metal catalyst, such as Pd on C under an atmposhere ofdeuterium gas.

Scheme F shows another general synthetic method for the preparation ofboronate intermediates where the aryl ring is substituted with adeuterium. A substituted 4-bromobenzenethiol is treated with a base suchas, but not limited to NaH, LiHMDS or KHMDS followed by quenching of theanion with deuterium source such as1,1,1,2,3,3,3-heptadeuterio-2-iodo-propane. The sulfide is then oxidizedto the corresponding sulfone using, for example, mCPBA or Oxone. Thehalogen is then transformed into a suitable boronate derivative via, forexample, metal mediated cross-coupling catalyzed by, for instance,Pd(^(t)Bu₃)₂ or Pd(dppf)Cl₂.DCM. The remaining substituent is thenconverted into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitbale metal catalyst, such as Pdon C under an atmposhere of deuterium gas.

Scheme G shows another general synthetic method for the preparation ofboronate intermediates where the aryl ring is substituted with adeuterium. A substituted 4-bromobenzenethiol is treated with a base suchas, but not limited to NaH, LiHMDS or KHMDS followed by quenching of theanion with for instance MeI. The sulfide is then oxidized to thecorresponding sulfone using, for example, mCPBA or Oxone. The sulfone istreated with a base such as, but not limited to NaH, LiHMDS or KHMDSfollowed by quenching of the anion with deuterium source such as D₃CI.This reaction is repeated until the desired amount of deuterium has beenincorporated into the molecule. The halogen is then transformed into asuitable boronate derivative via, for example, metal mediatedcross-coupling catalyzed by, for instance, Pd(^(t)Bu₃)₂ orPd(dppf)Cl₂.DCM. The remaining substituent is then converted intodeuterium by, for instance, metal catalyzed halogen-deuterium exchangeusing a suitbale metal catalyst, such as Pd on C under an atmposhere ofdeuterium gas.

Scheme H shows a general synthetic method for the preparation of oximeintermediates where the aryl ring is substituted with a deuterium. Themethyl group of an appropriately substituted methyl 4-methylbenzoatederivative can be converted into the corresponding dibromide underconditions such as AIBN catalyzed bromination with NBS. This di-bromideis then hydrolysed to the corresponding aldehyde, for instance usingAgNO₃ in acetone/water. Protection of the aldehyde as a suitable acetal,for instance the diethyl acetal and subsequent conversion of theremaining substituent into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitable metal catalyst, such as Pdon C under an atmosphere of deuterium gas gives the deuterated esterintermediate. The ester functionality can be reduced using reagents suchas LiAlH₄, NaBH₄, NaBD₄ or LiAlD₄ to give corresponding aldehyde. Thiscan be reacted under reductive amination conditions using a suitableamine, such as methylamine or d3-methylamine using a reducing agent suchas NaBH₄ or NaBD₄ to give the corresponding amine derivative. This canbe protected with, for instance a Boc group and the acetal convertedinto the oxime using, for instance, hydroxylamine hydrochloride inTHF/water.

Scheme I shows another general synthetic method for the preparation ofoxime intermediates where the aryl ring is substituted with a deuterium.The methyl group of an appropriately substituted methyl 4-methylbenzoatederivative can be converted into the corresponding dibromide underconditions such as AIBN catalyzed bromination with NBS. This di-bromideis then hydrolysed to the corresponding aldehyde, for instance usingAgNO₃ in acetone/water. Protection of the aldehyde as a suitable acetal,for instance the dimethyl acetal and subsequent conversion of theremaining subtituent into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitable metal catalyst, such as Pdon C under an atmosphere of deuterium gas gives the deuterated esterintermediate. The ester functionality can be converted into thecorresponding primary amide under standard conditions, such as heatingwith a solution of ammonia in methanol. The amide can be reduced to thecorresponding amine using reagents not limited to LiAlH₄ or LiAlD₄. Thiscan be protected with, for instance a Boc group. The carbamate NH can bealkylated under basic conditions using for instance NaH, LiHMDS or KHMDSfollowed by quenching of the anion with deuterium source such MeI orD₃CI. The acetal can be converted into the oxime using, for instance,hydroxylamine hydrochloride in THF/water.

Scheme J shows another general synthetic method for the preparation ofoxime intermediates where the aryl ring is substituted with a deuterium.The methyl group of an appropriately substituted methyl 4-methylbenzoatederivative can be converted into the corresponding dibromide underconditions such as AIBN catalyzed bromination with NBS. This di-bromideis then hydrolysed to the corresponding aldehyde, for instance usingAgNO₃ in acetone/water. Protection of the aldehyde as a suitable acetal,for instance the dimethyl acetal and subsequent conversion of theremaining substituent into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitable metal catalyst, such as Pdon C under an atmposhere of deuterium gas gives the deuterated esterintermediate. The ester functionality can be converted into thecorresponding primary amide under standard conditions, such as heatingwith a solution of ammonia in methanol. The amide can be reduced to thecorresponding amine using reagents not limited to LiAlH₄ or LiAlD₄. Thiscan be reacted under reductive amination conditions using a suitableamine, such as methylamine, d3-methylamine, formaldehyde ord2-formaldehyde using a reducing agent such as NaBH₄ or NaBD₄ to givethe corresponding amine derivative. This can be protected with, forinstance a Boc group. The acetal can be converted into the oxime using,for instance, hydroxylamine hydrochloride in THF/water.

Scheme K shows another general synthetic method for the preparation ofoxime intermediates where the aryl ring is substituted with a deuterium.The methyl group of an appropriately substituted methyl 4-methylbenzoatederivative can be converted into the corresponding dibromide underconditions such as AIBN catalyzed bromination with NBS. This di-bromideis then hydrolysed to the corresponding aldehyde, for instance usingAgNO₃ in acetone/water. Protection of the aldehyde as a suitable acetal,for instance the dimethyl acetal and subsequent conversion of theremaining substituent into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitable metal catalyst, such as Pdon C under an atmosphere of deuterium gas gives the deuterated esterintermediate. This can be reacted under reductive amination conditionsusing a suitable amine, such as ammonium hydroxide using a reducingagent such as NaBH₄ or NaBD₄ to give the corresponding amine derivative.This can be protected with, for instance a Boc group and the carbamateNH alkylated under basic conditions using for instance NaH, LiHMDS orKHMDS followed by quenching of the anion with deuterium source such Metor D₃CI. The ester can be reduced to the corresponding alcohol using asuitable reducing agent such as LiBH₄ or NaBH₄. The alcohol can beoxidized to the aldehyde using regeants such as MnO₂ or Dess-Martinperiodane. The acetal can be converted into the oxime using, forinstance, aqueous hydroxylamine.

Scheme L shows a general synthetic method for the preparation ofdeuterated oxime intermediates. 4-(diethoxymethyl)benzaldehyde can bereacted under reductive amination conditions using a suitable amine,such as methylamine or d3-methylamine using a reducing agent such asNaBH₄ or NaBD₄ to give the corresponding amine derivative. This can beprotected with, for instance a Boc group and the acetal converted intothe oxime using, for instance, hydroxylamine hydrochloride in THF/water.

Scheme M shows another general synthetic method for the preparation ofdeuterated oxime intermediates. The ester functionality of methyl4-(dimethoxymethyl)benzoate can be converted into the correspondingprimary amide under standard conditions, such as heating with a solutionof ammonia in methanol. The amide can be reduced to the correspondingamine using reagents not limited to LiAlH₄ or LiAlD₄. This can beprotected with, for instance a Boc group. The carbamate NH can bealkylated under basic conditions using for instance NaH, LiHMDS or KHMDSfollowed by quenching of the anion with deuterium source such MeI orD₃CI. The acetal can be converted into the oxime using, for instance,hydroxylamine hydrochloride in THF/water.

Scheme N shows another general synthetic method for the preparation ofdeuterated oxime intermediates. The ester functionality of methyl4-(dimethoxymethyl)benzoate can be converted into the correspondingprimary amide under standard conditions, such as heating with a solutionof ammonia in methanol. The amide can be reduced to the correspondingamine using reagents not limited to LiAlH₄ or LiAlD₄. This can bereacted under reductive amination conditions using a suitable amine,such as methylamine, d3-methylamine, formaldehyde or d2-formaldehydeusing a reducing agent such as NaBH₄ or NaBD₄ to give the correspondingamine derivative. This can be protected with, for instance a Boc group.The acetal can be converted into the oxime using, for instance,hydroxylamine hydrochloride in THF/water.

Scheme O shows another general synthetic method for the preparation ofdeuterated oxime intermediates. A 4-substituted benzylmine can beprotected with, for instance a Boc group. The carbamate NH can bealkylated under basic conditions using for instance NaH, LiHMDS or KHMDSfollowed by quenching of the anion with deuterium source such MeI orD₃CI. The ester can be reduced to the corresponding alcohol using asuitable reducing agent such as LiBH₄ or NaBH₄. The alcohol can beoxidized to the aldehyde using regeants such as MnO₂ or Dess-Martinperiodane. The acetal can be converted into the oxime using, forinstance, aqueous hydroxylamine.

Scheme P shows a general synthetic method for the preparation ofdeuterated pyrazine-isoxazole derrivatives. 3,5-Dibromopyrazin-2-amineis converted into the corresponding silyl-protected alkyne understandard Sonagashira conditions utilizing, for example, Pd(PPh₃)₄ andCuI as catalysts. The pyrazine NH₂ can then be protected as, for examplethe di-Boc derivative. Coupling of the pyrazine bromide with a boronate,for instance those outlined in Schemes 1 to 6 above, under standardSuzuki cross-coupling conditions followed by removal of the silylprotecting group give the desired alkyne intermediate. Oximes, such asthose outline in Schems 7 to 14 above, can be converted into thecorresponding chlorooximes using, for instance, NCS. The alkyne andchlorooxime intermediates can undergo a [3+2] cycloaaditon to givecorresponding isoxazole under standard conditions, for instance by theaddition of Et₃N. The Boc protecting groups can be removed under acicidconditions such as TFA in DCM or HCl in MeOH/DCM to give the deuteratedpyrazine isoxale derrivatives.

Scheme Q shows a general synthetic method for the preparation ofdeuterated isoxazole derrivatives. The pyrazine NH₂ and benzylamineamineNH can be protected under standard conditions using trifluoroaceticanhydride. Halogenation of the isoxazole ring with, for example NISfollowed by removal of the trifluoroacetate protecting group under basicconditions provides the desired halogenated interemdiates. The halogencan then converted into deuterium by, for instance, metal catalyzedhalogen-deuterium exchange using a suitbale metal catalyst, such as Pdon C under an atmposhere of deuterium gas.

Abbreviations

The following abbreviations are used:

-   ATP adenosine triphosphate-   Boc tert-butyl carbamate-   Cbz Carboxybenzyl-   DCM dichloromethane-   DMSO dimethyl sulfoxide-   Et₃N triethylamine-   2-MeTHF 2-methyltetrahydrofuran-   NMM N-Methyl morpholine-   DMAP 4-Dimethylaminopyridine-   TMS Trimethylsilyl-   MTBE methyl tertbutyl ether-   EtOAc ethyl acetate-   i-PrOAc isopropyl acetate-   IPAC isopropyl acetate-   DMF dimethylformamide-   DIEA diisopropylethylamine-   TEA triethylamine-   t-BuONa sodium tertbutoxide-   K₂CO₃ potassium carbonate-   PG Protecting group-   pTSA para-toluenesulfonic acid-   TBAF Tetra-n-butylammonium fluoride-   ¹HNMR proton nuclear magnetic resonance-   HPLC high performance liquid chromatography-   LCMS liquid chromatography-mass spectrometry-   TLC thin layer chromatography-   Rt retention time

SCHEMES AND EXAMPLES

The compounds of the disclosure may be prepared in light of thespecification using steps generally known to those of ordinary skill inthe art. Those compounds may be analyzed by known methods, including butnot limited to LCMS (liquid chromatography mass spectrometry) and NMR(nuclear magnetic resonance). The following generic schemes and examplesillustrate how to prepare the compounds of the present disclosure. Theexamples are for the purpose of illustration only and are not to beconstrued as limiting the scope of the invention in any way. ¹H-NMRspectra were recorded at 400 MHz using a Bruker DPX 400 instrument. Massspec. samples were analyzed on a MicroMass Quattro Micro massspectrometer operated in single MS mode with electrospray ionization.

Example 1 Synthesis of2-(4-(5-amino-6-(3-(4-((tetrahydro-2H-pyran-4-ylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)pyridin-2-yl)-2-methylpropanenitrile(Compound I-1)

Method 1:

To a solution of tetrahydropyran-4-amine (100 g, 988.7 mmol) in MeOH(3.922 L) was added 4-(diethoxymethyl)benzaldehyde (196.1 g, 941.6 mmol)over 2 min at RT. The reaction mixture was stirred at RT for 80 min,until the aldimine formation was complete (as seen by NMR). NaBH4 (44.49g, 1.176 mol) was carrefully added over 45 min, maintaining thetemperature between 24° C. and 27° C. by mean of an ice bath. After 75min at RT, the reaction has gone to completion. The reaction mixture wasquenched with 1M NaOH (1 L). The reaction mixture was partitionedbetween brine (2.5 L) and TBDME (4 L then 2×1 L). The organic phase waswashed with brine (500 mL) and concentrated in vacuo. The crude mixturewas redisolved in DCM (2 L). The aqueous phase was separated, theorganic phase was dried over MgSO4, filtered and concentrated in vacuoto give the title compound as a yellow oil (252.99 g, 91%).

Method 2:

A solution ofN-[[4-(diethoxymethyl)phenyl]methyl]tetrahydropyran-4-amine (252.99 g,862.3 mmol) and Boc anhydride (191.9 g, 202.0 mL, 879.5 mmol) in DCM(2.530 L) was cooled down to 3.3° C. Et3N (89.00 g, 122.6 mL, 879.5mmol) was added over 4 min, keeping the internal temperature below 5° C.The bath was removed 45 min after the end of the addition. And thereaction mixture was stirred at RT overnight. The reaction mixture wassequentially washed with 0.5 M citric acid (1 L), saturated NaHCO3solution (1 L) and brine (1 L). The organic phase was dried (MgSO4),filtered and concentrated in vacuo to give a colourless oil (372.38 g,110%). 1H NMR (400.0 MHz, DMSO); MS (ES+)

Method 3:

tert-butylN-[[4-(diethoxymethyl)phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate(372.38 g, 946.3 mmol) was dissolved in THF (5 L) and water (500 mL).Hydroxylamine hydrochloride (72.34 g, 1.041 mol) was added in oneportion and the reaction mixture was stirred overnight at RT. Thereaction mixture was partitioned between DCM (5 L) and water. Thecombined organic extract was washed with water (1L×2). The organic phasewas concentrated in vacuo to a volume of about 2L. The organic layer wasdried over MgSO4, filtered and concentrated in vacuo to give a stickycolourless oil that crystallized on standing under vacuo. (334.42g,106%). 1H NMR (400.0 MHz, CDCl3); MS (ES+)

Method 4:

tert-butylN-[[4-[(E)-hydroxyiminomethyl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate(334.13 g, 999.2 mmol) was dissolved in isopropyl acetate (3.0 L) (themixture was warmed to 40° C. to allow all the solids to go intosolution). N-chlorosuccinimide (140.1 g, 1.049 mol) was addedportionwise over 5 min and the reaction mixture was heated to 55° C.(external block temperature). After 45 min at 55° C. The reaction hadgone to completion. The reaction mixture was cooled down to RT. Thesolids were filtered off and rinsed with Isopropyl acetate (1 L).Combined organic extract was sequentially washed with water (1.5 L, 5times) and brine, dried over MgSO4, filtered and concentrated in vacuoto give a viscous yellow oil (355.9 g; 96%). 1H NMR (400.0 MHz, CDCl3);MS (ES+)

Method 5:

Et₃N (76.97 g, 106.0 mL, 760.6 mmol) was added over 20 minutes to asolution of tert-butylN-(5-bromo-3-ethynyl-pyrazin-2-yl)-N-tert-butoxycarbonyl-carbamate(233.0 g, 585.1 mmol) and tert-butylN-[[4-[(Z)—C-chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate(269.8 g, 731.4 mmol) in DCM (2.330 L) at RT. During addition oftriethylamine, the exotherm was stabilised by cooling the mixture in anice bath, then the reaction mixture was gradually warmed up to RT andthe mixture was stirred at RT overnight. The reaction mixture wassequentially washed with water (1.5 L, 3 times) and brine. The organicextract was dried over MgSO4, filtered and partially concentrated invacuo. Heptane (1.5L) was added and the concentration was continuedyielding 547.63 g of a yellow-orange solid.

542.12 g was taken up into ˜2 vol (1 L) of ethyl acetate. The mixturewas heated to 74-75° C. internally and stirred until all the solid wentinto solution. Heptane (3.2 L) was added slowly via addition funnel tothe hot solution keeping the internal temperature between 71° C. and 72°C. At the end of the addition, the dark brown solution was seeded withsome recrystallised product, and the reaction mixture was allowed tocool down to RT without any stirring to crystallise O/N. The solid wasfiltered off and rinsed with heptane (2×250 mL), then dried in vacuo toyield 307.38 g of the title product (72%). %). 1H NMR (400.0 MHz,CDCl3); MS (ES+)

Method 6:

tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-bromo-pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate (303 g,414.7 mmol) and2-methyl-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]propanenitrile (112.9 g, 414.7 mmol) were suspended in MeCN (2 L) andH₂O (1 L). Na₂CO₃ (414.7 mL of 2 M, 829.4 mmol) followed by Pd[P(tBu)3]2(21.19 g, 41.47 mmol) were added and the reaction mixture was degassedwith N2 for 1 h. The reaction mixture was placed under a nitrogenatmosphere and heated at 70° C. (block temperature) for 4 h (internaltemperature fluctuated between 60° C. and 61° C.). The reaction wascooled down to room temperature and stirred at RT overnight. Thereaction mixture was partitioned between EtOAc (2 L) and water (500 mL).The combined organic extract was washed with brine (500 mL), filteredthrough a short pad of celite and concentrated under reduced pressure toa volume of about 3 L. The solution was dried over MgSO4, filtered andpartially concentrated in vacuo. iPrOH (1.5 L) was added and the solventwas removed in vacuo to yield the desired product as a light brown foam(405 g).

400 g was taken up into ˜5 vol (2 L) of iPrOH and the mixture was heatedto 80° C. until all the solid went into solution. The dark brownsolution was seeded, and the reaction mixture was allowed to slowly cooldown to RT overnight. The solid was filtered off and rinsed with iPrOH(2×250 mL) and Petroleum ether (2×200 mL). The resulting solid wasslurried in petroleum ether (2.5 L), filtered off and dried in vacuo.The resulting solid was dissolved in DCM (2.5 L) and stirred slowly for1 h with 30 g of SPM32 (3-mercaptopropyl ethyl sulfide silica). Thesilica was filtered through a pad of florisil and rinsed with DCM. Theprocedure was repeated twice, then the DCM solution was concentrated invacuo to give 238.02 g of a light yellow solid.

Method 7:

tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-[2-(1-cyano-1-methyl-ethyl)-4-pyridyl]pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate(238 g, 299.0 mmol) was dissolved in DCM (2.380 L). TFA (500 mL, 6.490mol) was added at RT over 3 min. The reaction mixture was stirred at RTfor 3.5 h. The reaction mixture was concentrated under reduced pressurethen azeotroped with heptane (2×300 ml). The oil was then slurried inabs. EtOH (2.5 L) and filtered . The solid was dissolved in a mixture ofethanol (1.190 L) and water (1.190 L). potassium carbonate (124.0 g,897.0 mmol) in water (357.0 mL) was added to the solution and themixture was stirred at RT overnight.

The solid was filtered off, was washed with water (2.5 L), and dried at50° C. in vacuo to give 108.82 g of the title compound (Compound I-1) asa yellow powder. (73%)

Methods 6a and 7a

A mixture of tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-bromo-pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate (110.0 g,151 mmol), K₂CO₃ (41.6 g, 301 mmol), and2-methyl-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]propanenitrile (41.0 g, 151 mmol) in toluene (770 mL) and water (220 mL)is stirred and degassed with N₂ for 30 min. at 20° C. The catalystPd(dtbpf)Cl₂ (1.96 g, 3.01 mmol) is added and the mixture is degassedfor an additional 10 min. The mixture is heated at 70° C. until thereaction is complete. The mixture is cooled to ambient temperature,diluted with water (220 mL), and filtered through a bed of Celite. Theorganic phase is concentrated to remove most of the solvent. Theconcentrate is diluted with i-PrOH (550 mL). The resultant suspension isstirred for at least 1 h and then the solid is collected by filtrationto afford a tan powder. The solid is dissolved in toluene (990 mL) andstirred with Biotage MP-TMT resin (18.6 g) for 2 h at ambienttemperature. The resin is removed by filtration. The filtrate isconcentrated then diluted with i-PrOH (550 mL) and then re-concentratd.Add i-PrOH (550 mL) and stir for 1 h at ambient temperature. Cool thesuspension to 5° C. and collect the solid by filtration then dry toafford tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-[2-(1-cyano-1-methyl-ethyl)-4-pyridyl]pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate(Compound I-1) (81.9 g; 68%, yield, 98.7 area % purity by HPLC) as acream-colored powder.

Form Change to Compound I-1.HCl.1.5 H₂O

A suspension of tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-[2-(1-cyano-1-methyl-ethyl)-4-pyridyl]pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-tetrahydropyran-4-yl-carbamate(Compound I-1) (36.0 g, 72.6 mmol) in CH₃CN (720 mL) is stirred atambient temperature (20° C.) in a flask equipped with mechanicalstirring. A 1 M aqueous solution of HCl (72.6 mL; 72.6 mmol) is added.The suspension is stirred at ambient temperature for 20 h. The solid iscollected by filtration. The filter-cake is washed with CH₃CN (3×50 mL)then dried under vacuum with high humidity for 2 h to afford CompoundI-1.HCl.1.5 H₂O (30.6 g; 74%) yield, 98.8 area % purity by HPLC) as ayellow powder. ¹H NMR (400 MHz, DMSO) δ 9.63 (d, J=4.7 Hz, 2H), 9.05 (s,1H), 8.69 (d, J=5.2 Hz, 1H), 8.21 (s, 1H), 8.16-8.03 (m, 3H), 7.84 (t,J=4.1 Hz, 3H), 7.34 (br s, 2H), 4.40-4.18 (m, 2H), 3.94 (dd, J=11.2, 3.9Hz, 2H), 3.32 (t, J=11.2 Hz, 3H), 2.17-2.00 (m, 2H), 1.81 (s, 6H), 1.75(dd, J=12.1, 4.3 Hz, 2H).

Example 2 Synthesis of3-[3-[4-[dideuterio(methylamino)methyl]phenyl]isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-amine(Compound II-1)

Step 1: 5-Bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-amine

(Trimethylsilyl)acetylene (1.845 g, 2.655 mL, 18.78 mmol) was addeddropwise to a solution of 3,5-dibromopyrazin-2-amine (compound i) (5 g,19.77 mmol) in DMF (25 mL). Triethylamine (10.00 g, 13.77 mL, 98.85mmol), copper(I) iodide (451.7 mg, 2.372 mmol) and Pd(PPh₃)₄ (1.142 g,0.9885 mmol) were then added and the resulting solution stirred at RTfor 30 minutes.The reaction mixture was diluted with EtOAc and water andthe layers separated. The aqueous layer was extracted further with EtOAcand the combined organic layers washed with water, dried (MgSO₄) andconcentrated in vacuo. The residue was purified by column chromatographyeluting with 15% EtOAc/Petroleum ether to give the product as a yellowsolid (3.99 g, 75% Yield). 1H NMR (400.0 MHz, DMSO) δ 0.30 (9H, s), 8.06(IH, s); MS (ES+) 271.82.

Step 2: tert-ButylN-tert-butoxycarbonyI-N-[5-bromo-3-((trimethylsilyl)ethyynyl)pyrazin-2-yl]carbamate

5-Bromo-3-(2-trimethylsilylethynyl)pyrazin-2-amine (2.85 g, 10.55 mmol)was dissolved in DCM (89.06 mL) and treated with Boc anhydride (6.908 g,7.272 mL, 31.65 mmol) followed by DMAP (128.9 mg, 1.055 mmol). Thereaction was allowed to stir at ambient temperature for 2 hours. Themixture was then diluted with DCM and NaHCO₃ and the layers separated.The aqueous layer was extracted further with DCM, dried (MgSO₄),filtered and concentrated in vacuo. The resultant residue was purifiedby column chromatography eluting with dichloromethane to give thedesired product as a colourless oil (4.95g, 99% Yield). 1H NMR (400.0MHz, DMSO) δ 0.27 (9H, s), 1.42 (18H, s), 8.50 (1H, s); MS (ES+) 472.09.

Step 3: tert-ButylN-(3-ethynyl-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)N-tertbutoxycarbonyl-carbamatetert-butyl

N-[5-Bromo-3-(2-trimethylsilylethynyl)pyrazin-2-yl]-N-tertbutoxycarbonylcarbamate(3 g, 6.377 mmol) and (4-isopropylsulfonylphenyl)boronic acid (1.491 g,6.536 mmol) were dissolved in MeCN/water (60/12 mL). K₃PO₄ (2.706 g,12.75 mmol) was added and the reaction mixture was degassed with a flowof nitrogen (5 cycles). Pd[P(tBu)₃]₂ (162.9 mg, 0.3188 mmol) was addedand the resulting mixture was stirred at room temperature for 1 h. Thereaction mixture was poured quickly into a mixture of ethyl acetate (500mL), water (90 mL) and 1% aqueous sodium metabisulphite at 4° C., shakenwell and the layer separated. The organic fraction was dried over MgSO₄,filtered and the filtrate was treated with 3-mercaptopropyl ethylsulphide on silica (0.8 mmol/g, 1 g), pre-absorbed onto silica gel thenpurified by column chromatography on silica gel eluting with 30-40%EtOAc/petroleum ether. The solvents were concentrated in vacuo to leavethe product as a yellow viscous oil that was triturated with petroleumether to yield the product as beige crystals (1.95 g, 61% Yield); 1H NMR(400 MHz, DMSO) δ 1.20 (m, 6H), 1.39 (s, 18H), 3.50 (m, 1H), 5.01 (s,1H), 8.03 (m, 2H), 8.46 (m, 2H) and 9.37 (s, IH).

Step 4: 4-(Dimethoxymethyl)benzamide

A mixture of methyl 4-(dimethoxymethyl)benzoate (3.8 g, 18.08 mmol) and7M NH₃ in MeOH (30 mL of 7 M, 210.0 mmol) in a sealed tube was heated at110° C. for 22 hours. A further portion of 7M NH₃ in MeOH (20 mL of 7 M,140.0 mmol) was added and the reaction heated at 135° C. for 23 hours.The reaction was cooled to ambient temperature and the solvent removedin vacuo. The residue was re-submitted to the reaction conditions (7MNH₃ in MeOH (30 mL of 7 M, 210.0 mmol) at 115° C.) for a further 16hours. The solvent was removed in vacuo and the residue tritruated fromEt₂O. The resultant precipitate was isolated by filtration to give thesub-title compound as a white solid (590 mg, 17% yield). The filtratewas purified by column chromatography (ISCO Companion, 40 g column,eluting with 0 to 100% EtOAc/Petroleum Ether to 10% MeOH/Et0Ac, loadedin EtOAc/MeOH) to give a further protion of the sub-title product as awhite solid (225 mg, 6% Yield). Total isolated (815 mg, 23% Yield); 1HNMR (400 MHz, DMSO) δ 3.26 (s, 6H), 5.44 (s, 1H), 7.37 (s, 1H), 7.46 (d,J=8.0 Hz, 2H), 7.84-7.91 (m, 2H) and 7.98 (s, 1H) ppm; MS (ES+) 196.0.

Step 5: Dideuterio-[4-(dimethoxymethyl)phenyl]methanamine

LiDH₄ (12.52 mL of 1 M, 12.52 mmol) was added dropwise to a stirredsolution of 4-(dimethoxymethyl)benzamide (815 mg, 4.175 mmol) in THF (20mL) at 0° C. under an atmosphere of nitrogen. The reaction was heated atreflux for 16 hours then cooled to ambient temperature. The reaction wasquenched by the sequential addition of D₂O (1 mL), 15% NaOH in D₂O (1mL) and D₂O (4 mL). The resultant solid was removed by filtration andwashed with EtOAc. The filtrate was concentrated in vacuo and theresidue dried by azeotropic distillation with toluene (×3) to give thesub-title compound as a yellow oil (819 mg) that was used withoutfurther purification; 1H NMR (400 MHz, DMSO) δ 3.23 (s, 6H), 5.36 (s,1H) and 7.30-7.35 (m, 4H) ppm; MS (ES+) 167.0.

Step 6: tert-ButylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]carbamate

Et₃N (633.7 mg, 872.9 μL, 6.262 mmol) was added to a stirred suspensionof dideuterio-[4-(dimethoxymethyl)phenyl]methanamine (765 mg, 4.175mmol) in THF (15 mL) at 0° C. The reaction was allowed to stir at thistemperature for 30 minutes then Boc₂O (956.8 mg, 1.007 mL, 4.384 mmol)was added in portions. The reaction was allowed to warm to ambienttemperature and stirred for 18 hours. The solvent was removed in vacuoand the residue was purified by column chromatography (ISCO Companion,120 g column, eluting with 0 to 50% EtOAc/Petroleum Ether, loaded inDCM) to give the sub-title product as a colourless oil (1.04 g, 88%Yield); 1H NMR (400 MHz, DMSO) δ 1.40 (s, 9H), 3.23 (s, 6H), 5.36 (s,1H), 7.24 (d, J=8.2 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H) and 7.38 (s, 1H)ppm.

Step 7: tert-ButylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]-N-methyl-carbamate

LiHMDS (1M in THF) (1.377 mL of 1 M, 1.377 mmol) was added dropwise to asittred solution of tert-butylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]carbamate (300 mg, 1.059mmol) in THF (5 mL) at −78° C. The solution was stirred at thistemperature for 10 minutes then iodomethane (225.4 mg, 98.86 μL, 1.588mmol) was added dropwise and the mixture allowed to warm to ambienttemperature over 1 hour. The reaction was again cooled to −78° C. andLiHMDS (1M in THF) (635.4 μL of 1 M, 0.6354 mmol) was added. After 10minutes iodomethane (105.2 mg, 46.14 μL, 0.7413 mmol) was added and thereaction allowed to warm to ambient temperature over 6 hours. Themixture was diluted with EtOAc and the organic layer washed withsaturated aqueous NaHCO₃ (×2), brine (×1), dried (MgSO₄) filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion, 24 g column, eluting with 0 to 30% EtOAc/PetroleumEther, loaded in DCM) to give the sub-title product as a colourless oil(200 mg, 63% Yield); 1H NMR (400 MHz, DMSO) δ 1.41 (d, J=27.7 Hz, 9H),2.76 (s, 3H), 3.24 (s, 6H), 5.37 (s, 1H), 7.23 (d, J=7.9 Hz, 2H) and7.37 (d, J=8.0 Hz, 2H) ppm.

Step 8: tert-ButylN-[dideuterio-[4-[hydroxyiminomethyl]phenyl]methyl]-N-(methyl)carbamate

Hydroxylamine hydrochloride (51.15 mg, 0.7361 mmol) was added to astirred solution of tert-butylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]-N-methyl-carbamate (199mg, 0.6692 mmol) in THF (10 mL)/water (1.000 mL) and the reactionallowed to stir at ambient temperature for 4 hours. The reaction waspartitioned between DCM and brine and the layers separated. The aqueouslayer was extracted with DCM (×2) and the combined organic extractswashed with brine (×1), dried (MgSO₄), filtered and concentrated invacuo to give the sub-title compound as a white solid (180 mg, 100%Yield); 1H NMR (400 MHz, DMSO) δ 1.41 (d, J=24.6 Hz, 9H) 2.76 (s, 3H),7.25 (d, J=8.1 Hz, 2H), 7.58 (d, J=8.0 Hz, 2H), 8.13 (s, 1H) and 11.20(s, 1H) ppm; MS (ES+) 211.0 (M-Boc).

Step 9: tert-ButylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]-dideuterio-methyl]-N-methyl-carbamate

tert-ButylN-[dideuterio-[4-[hydroxyiminomethyl]phenyl]methyl]-N-(methyl)carbamate(178 mg, 0.6683 mmol) in DMF (2 mL) was treated with NCS (89.24 mg,0.6683 mmol) and the reaction warmed to 65° C. for 1 hour. The reactionwas cooled to ambient temperature and diluted with water. The mixturewas extracted with EtOAc (×2) and the combined organic extracts washedwith brine (×4), dried (MgSO₄), filtered and concentrated in vacuo togive the sub-title compound as a white solid (188 mg, 94% Yield); 1H NMR(400 MHz, DMSO) δ 1.42 (d, J=24.7 Hz, 9H), 2.78 (s, 3H), 7.32 (d, J=8.4Hz, 2H), 7.78 (d, J=8.2 Hz, 2H) and 12.36 (s, 1H) ppm.

Step 10: tert-ButylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]-dideuterio-methyl]-N-methyl-carbamate

Et₃N (36.31 mg, 50.01 μL, 0.3588 mmol) was added dropwise to a stirredsolution of tert-butylN-tert-butoxycarbonyl-N-[3-ethynyl-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]carbamate(150 mg, 0.2990 mmol) and tert-ButylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]-dideuterio-methyl]-N-methyl-carbamate(89.93 mg, 0.2990 mmol) in anhydrous THF (3 mL) and the reaction mixtureheated at 65° C. for 3 hours. The reaction mixture was cooled to ambienttemperature and diluted with EtOAc/brine. Water was added until theaqueous layer became clear and the layers were separated. The aqueouslayer was extracted with EtOAc (×1) and the combined organic extractswere washed with brine (×1), dried (MgSO₄), filtered and concentrated invacuo. The residue was purified by column chromatography (ISCOCompanion, 40 g column, elueting with 0 to 30% EtOAc/Petroleum Ether,loaded in DCM) to give the sub-title product as a white solid (134 mg,59% Yield); 1H NMR (400 MHz, DMSO) δ 1.22 (d, J=6.8 Hz, 6H) 1.32 (s,18H), 1.43 (d, J=23.1 Hz, 9H), 2.82 (s, 3H), 3.56 (pent, 1H), 7.43 (d,J=8.3 Hz, 3H), 8.02-8.03 (m 3H), 8.06-8.11 (m, 2H), 8.62-8.67 (m, 2H)and 9.51 (s, 1H) ppm; MS (ES+) 666.2 (M-Boc).

Step 11:3-[3-[4-[Dideuterio(methylamino)methyl]phenyl]isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-amine(compound II-1)

3M HCl in MeOH (1.167 mL of 3 M, 3.500 mmol) was added to a stirredsolution of tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]-dideuterio-methyl]-N-methyl-carbamate(134 mg, 0.1750 mmol) in DCM (5 mL) and the reaction heated at refluxfor 16 hours. The reaction was cooled to ambient temperature and theresultant precipitate was isolated by filtration and dried under vacuumat 40° C. to give the di-HCl salt of the title compound as a yellowsolid (58.8 mg, 62% Yield); 1H NMR (400 MHz, DMSO) δ 1.20 (d, J=6.8 Hz,6H), 2.60 (t, J=5.4 Hz, 3H), 3.48 (hept, J=6.8 Hz, 1H), 7.22 (br s, 2H),7.69-7.75 (m, 2H), 7.85 (s, 1H), 7.92-7.99 (m, 2H), 8.08-8.15 (m, 2H)8.37-8.42 (m, 2H), 8.97 (s, 1H) and 9.10 (d, J=5.8 Hz, 2H) ppm; MS (ES+)466.2.

Example 3 Synthesis of3-[3-[4-[dideuterio-(trideuteriomethylamino)methyl]phenyl]isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-amine(Compound II-2)

Step 1: tert-ButylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]-I-(trideuteriomethyl)carbamate

LiHMDS (1M in THF) (1.181 mL of 1 M, 1.181 mmol) was added dropwise to asittred solution of tert-butylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]carbamate (300 mg, 1.059mmol) in THF (5 mL) at −78° C. The solution was stirred at thistemperature for 30 minutes then trideuterio(iodo)methane (198.0 mg,84.98 μL, 1.366 mmol) was added dropwise and the mixture allowed to warmto ambient temperature over 21 hours. The reaction was again cooled to−78° C. and a further portion of LiHMDS (1M in THF) (635.4 μL of 1 M,0.6354 mmol) was added. After 15 minutes more trideuterio(iodo)methane(76.75 mg, 32.94 μL, 0.5295 mmol) was added and the reaction allowed towarm to ambient temperature over 5 hours. The mixture was diluted withEtOAc and the organic layer washed with saturated aqueous NaHCO₃ (×2),brine (×1), dried (MgSO₄) filtered and concentrated in vacuo. Theresidue was purified by column chromatography (ISCO Companion, 24 gcolumn, eluting with 0 to 30% EtOAc/Petroleum Ether, loaded in DCM) togive the sub-title product as a colourless oil (213 mg, 67% Yield); 1HNMR (400 MHz, DMSO) δ 1.36-1.42 (m, 9H) 3.22 (s, 6H), 5.35 (s, 1H), 7.21(d, J=7.8 Hz, 2H) and 7.35 (d, J=7.7 Hz, 2H) ppm.

Step 2: tert-ButylN-[dideuterio-[4-[hydroxyiminomethyl]phenyl]methyl]-N-(trideuteriomethyl)carbamate

Hydroxylamine hydrochloride (53.95 mg, 0.7763 mmol) was added to astirred solution of tert-butylN-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]-N-(trideuteriomethyl)carbamate(212 mg, 0.7057 mmol) in THF (10 mL)/water (1.000 mL) and the reactionallowed to stir at ambient temperature for 22 hours. The reaction waspartitioned between DCM and brine and the layers separated. The aqueouslayer was extracted with DCM (×2) and the combined organic extractswashed with brine (×1), dried (MgSO₄), filtered and concentrated invacuo to give the sub-title compound as a white solid (190 mg, 100%Yield).); 1H NMR (400 MHz, DMSO) δ 1.41 (d, J=24.2 Hz, 9H)), 7.25 (d,J=8.1 Hz, 2H), 7.58 (d, J=8.0 Hz, 2H), 8.13 (s, 1H) and 11.20 (s, 1H)ppm.

Step 3: tert-ButylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]-dideuterio-methyl]-N-(trideuteriomethyl)carbamate

tert-ButylN-[dideuterio-[4-[hydroxyiminomethyl]phenyl]methyl]-N-(trideuteriomethyl)carbamate(190.0 mg, 0.7054 mmol) in DMF (2 mL) was treated with NCS (94.19 mg,0.7054 mmol) and the reaction warmed to 65° C. for 1 hour. The reactionwas cooled to ambient temperature and diluted with water. The mixturewas extracted with EtOAc (×2) and the combined organic extracts washedwith brine (×4), dried (MgSO₄), filtered and concentrated in vacuo togive the sub-title compound as a white solid (198 mg, 93% Yield); 1H NMR(400 MHz, DMSO) δ 1.41 (d, J=26.0 Hz, 9H), 7.32 (d, J=8.3 Hz, 2H), 7.78(d, J=8.2 Hz, 2H) and 12.36 (s, 1H) ppm.

Step 4: tert-ButylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]-dideuterio-methyl]-N-(trideuteriomethyl)carbamate

Et₃N (36.31 mg, 50.01 μL, 0.3588 mmol) was added dropwise to a stirredsolution of tert-butylN-tert-butoxycarbonyl-N-[3-ethynyl-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]carbamate(150 mg, 0.2990 mmol) and tert-butylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]-dideuterio-methyl]-N-(trideuteriomethyl)carbamate(90.84 mg, 0.2990 mmol) in anhydrous THF (3 mL) and the reaction mixtureheated at 65° C. for 3.5 hours. The reaction mixture was cooled toambient temperature and diluted with EtOAc/brine. Water was added untilthe aqueous layer became clear and the layers were separated. Theaqueous layer was extracted with EtOAc (×1) and the combined organicextracts were washed with brine (×1), dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion, 40 g column, elueting with 0 to 35% EtOAc/PetroleumEther, loaded in DCM) to give the sub-title product as a white solid(158 mg, 69% Yield); 1H NMR (400 MHz, DMSO) δ 1.22 (d, J=6.8 Hz, 6H)),1.44 (d, J=22.0 Hz, 9H), 3.56 (dt, J=13.5, 6.7 Hz, 2H), 7.43 (d, J=8.2Hz, 3H), 8.02 (d, J=6.9 Hz, 2H), 8.08 (d, J=8.7 Hz, 2H), 8.65 (d, J=8.8Hz, 2H) and 9.51 (s, 1H) ppm; MS (ES+) 669.3 (M-Boc).

Step 5:3-[3-[4-[dideuterio-(trideuteriomethylamino)methyl]phenyl]isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-amine(Compound II-2)

3M HCl in MeOH (1.361 mL of 3 M, 4.084 mmol) was added to a stirredsolution of tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]-dideuterio-methyl]-N-(trideuteriomethyl)carbamate(157 mg, 0.2042 mmol) in DCM (5 mL) and the reaction heated at refluxfor 16 hours. The reaction was cooled to ambient temperature and theresultant precipitate was isolated by filtration and dried under vacuumat 40° C. to give the di-HCl salt of the title compound as a yellowsolid (72.5 mg, 66% Yield); 1H NMR (400 MHz, DMSO) δ 1.20 (d, J=6.8 Hz,6H), 3.48 (dq, J=13.6, 6.7 Hz, 1H), 7.21 (s, 2H), 7.68-7.78 (m, 2H),7.85 (s, 1H), 7.91-7.99 (m, 2H), 8.08-8.13 (m, 2H), 8.36-8.42 (m, 2H),8.96 (s, 1H) and 9.14 (s, 2H) ppm; MS (ES+) 469.1.

Example 4 Synthesis of5-(4-isopropylsulfonylphenyl)-3-[3-[4-[(trideuteriomethylamino)methyl]phenyl]isoxazol-5-yl]pyrazin-2-amine(Compound II-3)

Step 1: Methyl 4-[(tert-butoxycarbonylamino)methyl]benzoate

Et₃N (1.882 g, 2.592 mL, 18.60 mmol) was added to a stirred suspensionof methyl 4-(aminomethyl)benzoate (Hydrochloric Acid (1)) (1.5 g, 7.439mmol) in THF (20 mL) at 0° C. The reaction was allowed to stir at thistemperature for 30 minutes then Boc₂O (1.705 g, 1.795 mL, 7.811 mmol)was added in portions. The reaction was allowed to warm to ambienttemperature and stirred for 18 hours. The mixture was diluted withEtOAc. The organic layer was washed with 1M aqueous HCl (×2), saturatedaqueous NaHCO₃ (×2) and brine (×1). The organic layer was dried (MgSO₄),filtered and concentrated in vacuo to give the sub-title compound as awhite solid that was used without further purification (1.93 g, 98%Yield); 1H NMR (400 MHz, DMSO) δ 1.40 (s, 9H), 3.85 (s, 3H), 4.20 (d,J=6.1 Hz, 2H), 7.38 (d, J=8.2 Hz, 2H), 7.49 (t, J=6.1 Hz, 1H) and 7.92(d, J=8.2 Hz, 2H) ppm; MS (ES+) 251.1 (M-Me).

Step 2: Methyl4-[[tert-butoxycarbonyl(trideuteriomethyl)amino]methyl]benzoate

LiHMDS (1M in THF) (8.112 mL of 1 M, 8.112 mmol) was added dropwise to astirred solution of methyl 4-[(tert-butoxycarbonylamino)methyl]benzoate(1.93 g, 7.275 mmol) in THF (10 mL) at −78° C. The solution was stirredat this temperature for 30 minutes then trideuterio(iodo)methane (1.360g, 9.385 mmol) was added dropwise and the mixture allowed to warm toambient temperature over 3 hours. The reaction was cooled to −78° C. anda further portion of LiHMDS (1M in THF) (2.182 mL of 1 M, 2.182 mmol)was added. After 10 minutes a further portion oftrideuterio(iodo)methane (527.4 mg, 3.638 mmol) was added and thereaction allowed to warm to ambient temperature over 17 hours. Themixture was diluted with EtOAc and the organic layer washed withsaturated aqueous NaHCO₃ (×2), brine (×1), dried (MgSO₄) filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion, 120 g column, eluting with 0 to 30% EtOAc/PetroleumEther, loaded in DCM) to give the sub-title product as a pale yellow oil(1.37 g, 67% Yield); 1H NMR (400 MHz, DMSO) δ 1.38 (d, J=44.2 Hz, 9H),3.83 (s, 3H), 4.43 (s, 2H), 7.33 (d, J=8.2 Hz, 2H) and 7.94 (d, J=8.1Hz, 2H) ppm; MS (ES+) 268.1 (M-Me)

Step 3: tert-ButylN-[[4-(hydroxymethyl)phenyl]methyl]-N-(trideuteriomethyl)carbamate

LiBH₄ (158.5 mg, 7.278 mmol) was added to a stirred solution of methyl4-[[tert-butoxycarbonyl(trideuteriomethyl)amino]methyl]benzoate (1.37 g,4.852 mmol) in THF (10 mL) and the reaction warmed to 85° C. for 15hours. A further portion of LiBH₄ (158.5 mg, 7.278 mmol) was added andthe reaction stirred at 65° C. for a further 7 hours. The reactionmixture was cooled to ambient temperature then poured onto crushed iceand whilst stirring, 1M HCl was added dropwise until no effervescencewas observed. The mixture was stirred for 10 minutes then saturatedaqueous NaHCO₃ was added until the mixture was at pH 8. The aqueouslayer was extracted with EtOAc (×3) and the combined organic extractsdried (MgSO₄), filtered and concentrated in vacuo. The residue waspurified by column chromatography (ISCO Companion, 120 g column,elueting with 0 to 100% EtOAc/Petroleum Ether, loaded in DCM) to givethe sub-title product as a colourless oil (1.03 g, 84% Yield); 1H NMR(400 MHz, DMSO) δ 1.42 (d, J=14.6 Hz, 9H), 4.35 (s, 2H), 4.48 (d, J=5.7Hz, 2H), 5.15 (t, J=5.7 Hz, 1H), 7.18 (d, J=7.9 Hz, 2H) and 7.30 (d,J=7.7 Hz, 2H) ppm; MS (ES+) 181.1 (M-O^(t)Bu).

Step 4: tert-ButylN-[(4-formylphenyl)methyl]-N-(trideuteriomethyl)carbamate

MnO₂ (5.281 g, 1.051 mL, 60.75 mmol) was added to a stirred solution oftert-butylN-[[4-(hydroxymethyl)phenyl]methyl]-N-(trideuteriomethyl)carbamate (1.03g, 4.050 mmol) in DCM (10 mL) and the reaction stirred at ambienttemperature for 20 hours. The reaction was filtered through a pad ofCelite and washed with DCM. The filtrate was concentrated in vacuo togive the sub-title compound as a colourless oil (891 mg, 88% Yield); 1HNMR (400 MHz, DMSO) δ 1.40 (d, J=43.4 Hz, 9H), 4.48 (s, 2H), 7.43 (d,J=8.0 Hz, 2H), 7.91 (d, J=7.9 Hz, 2H) and 10.00 (s, 1H), ppm.

Step 5: tert-butylN-[[4-[hydroxyiminomethyl]phenyl]methyl]-N-(trideuteriomethyl)carbamate

Hydroxylamine (466.0 μL of 50% w/v, 7.054 mmol) was added to a stirredsolution of tert-butylN-[(4-formylphenyl)methyl]-N-(trideuteriomethyl)carbamate (890 mg, 3.527mmol) in ethanol (5 mL) and the reaction mixture stirred at ambienttemperature for 45 minutes. The reaction mixture was concentrated invacuo and the residue taken up in water and extracted with EtOAc (×3).The combined organic extracts were washed with brine (×1), dried(MgSO₄), filtered and concentrated in vacuo. The residue was trituratedfrom petroleum ether and the precipitate isolated by filtration to givethe sub-title product as a white solid (837 mg, 89% Yield); 1H NMR (400MHz, DMSO) δ 1.41 (d, J=25.8 Hz, 9H), 4.38 (s, 2H), 7.24 (d, J=8.0 Hz,2H), 7.58 (d, J=8.0 Hz, 2H), 8.13 (s, 1H) and 11.20 (s, 1H) ppm; MS(ES+) 212.0 (M-^(t)Bu).

Step 6: tert-ButylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-N-(trideuteriomethyl)carbamate

tert-butylN-[[4-[hydroxyiminomethyl]phenyl]methyl]-N-(trideuteriomethyl)carbamate(250 mg, 0.9351 mmol) in DMF (2.5 mL) was treated with NCS (124.9 mg,0.9351 mmol) and the reaction warmed to 65° C. for 1 hour. The reactionwas cooled to ambient temperature and diluted with water. The mixturewas extracted with EtOAc (×2) and the combined organic extracts washedwith brine (×4), dried (MgSO₄), filtered and concentrated in vacuo togive the sub-title compound as a white solid (259 mg, 92% Yield); 1H NMR(400 MHz, DMSO) δ 1.41 (d, J=29.6 Hz, 9H), 4.42 (s, 2H), 7.31 (d, J=8.3Hz, 2H), 7.78 (d, J=8.0 Hz, 2H), and12.38 (s, 1H), ppm.

Step 7: tert-ButylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-(trideuteriomethyl)carbamate

Et₃N (48.41 mg, 66.68 μL, 0.4784 mmol) was added dropwise to a stirredsolution of tert-butylN-tert-butoxycarbonyl-N-[3-ethynyl-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]carbamate(200 mg, 0.3987 mmol) and tert-butylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-N-(trideuteriomethyl)carbamate(120.3 mg, 0.3987 mmol) in anhydrous THF (5 mL) and the reaction mixtureheated at 65° C. for 2.5 hours. The reaction mixture was cooled toambient temperature and diluted with EtOAc/brine. Water was added untilthe aqueous layer became clear and the layers were separated. Theaqueous layer was extracted with EtOAc (×1) and the combined organicextracts were washed with brine (×1), dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion, 40 g column, elueting with 0 to 20% EtOAc/PetroleumEther, loaded in DCM) to give the sub-title product as a white solid(213.5 mg, 70% Yield); 1H NMR (400 MHz, DMSO) δ 1.22 (d, J=6.8 Hz, 6H),1.31 (s, 18H), 1.43 (d, J=26.2 Hz, 9H), 3.51-3.60 (m, 1H), 4.47 (s, 2H),7.42 (d, J=8.1 Hz, 2H), 8.03 (d, J=5.2 Hz, 3H), 8.08 (d, J=8.6 Hz, 2H),8.65 (d, J=8.6 Hz, 2H) and 9.52 (s, 1H) ppm; MS (ES+) 667.4 (M-Boc).

Step 8:5-(4-Isopropylsulfonylphenyl)-3-[3-[4-[(trideuteriomethylamino)methyl]phenyl]isoxazol-5-yl]pyrazin-2-amine(Compound II-3)

3M HCl in MeOH (1.5 mL of 3 M, 4.500 mmol) was added to a stirredsolution of tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-(trideuteriomethyl)carbamate(213 mg, 0.2777 mmol) in DCM (6 mL) and the reaction heated at refluxfor 15 hours. A further portion of 3M HCl in MeOH (0.5 mL of 3 M, 1.500mmol) was added and the reaction heated at reflux for a further 7 hours.The reaction was cooled to ambient temperature and the resultantprecipitate was isolated by filtration and dried under vacuum at 40° C.to give the di-HCl salt of the title compound as a yellow solid (97.6mg, 65% Yield); 1H NMR (400 MHz, DMSO) δ 1.20 (d, J=6.8 Hz, 6H), 3.47(tt, J=14.0, 6.9 Hz, 1H), 4.19-4.25 (m, 2H), 7.23 (s, 2H), 7.72 (d,J=8.4 Hz, 2H), 7.85 (s, 1H), 7.95 (d, J=8.7 Hz, 2H), 8.11 (d, J=8.4 Hz,2H), 8.39 (d, J=8.7 Hz, 2H), 8.97 (s, 1H) and 9.11 (s, 2H) ppm; MS (ES+)467.2.

Example 5 Synthesis of3-[3-[4-(Methylaminomethyl)phenyl]isoxazol-5-yl]-5-[4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonylphenyl]pyrazin-2-amine(Compound II-4)

Step 1: 1-[4-(Diethoxymethyl)phenyl]-N-methyl-methanamine

2M methylamine in MeOH (288.1 mL, 576.2 mmol) was diluted with methanol(1.000 L) and stirred at ˜20° C. 4-(Diethoxymethyl)benzaldehyde (100 g,480.2 mmol) was added dropwise over 1 minute and the reaction stirred atambient temperature for 1.25 hours. Sodium borohydride (29.07 g, 30.76mL, 768.3 mmol) was added portionwise over 20 minutes while maintainingthe temperature between 20 and 30° C. with an ice-water bath. Thereaction solution was stirred at ambient temperature overnight thenquenched by the dropwise addition of NaOH (960.4 mL of 1.0 M, 960.4mmol) over 20 minutes. The reaction was stirred for 30 minutes andconcentrated in vacuo to remove MeOH. The reaction was partitioned withMTBE (1.200 L) and the phases separated. The organic phase was washedwith water (300.0 mL), dried (Na₂SO₄), and concentrated in vacuo to givethe title compound as a yellow oil (102.9 g, 96% Yield); 1H NMR (400MHz, CDCl₃) δ 1.25 (t, 6H), 2.46 (s, 3H), 3.45-3.65 (m, 4H), 3.75 (s,2H), 5.51 (s, 1H), 7.32 (d, 2H) and 7.44 (d, 2H) ppm.

Step 2: tert-ButylN-[[4-(diethoxymethyl)phenyl]methyl]-N-methyl-carbamate

A 1-L glass jacketed reactor was fitted with an overhead stirrer,thermocouple, and chiller. A solution of1-[4-(diethoxymethyl)phenyl]-N-methyl-methanamine (80.0 g, 358.2 mmol)in DCM (480.0 mL) was stirred at 18° C. A solution of Boc anhydride(79.75 g, 83.95 mL, 365.4 mmol) in DCM (160.0 mL) was added over 10minutes and the solution was stirred at 20-25° C. overnight. Thereaction mixture was filtered, rinsed with DCM (3×50 mL) and thefiltrate concentrated in vacuo to afford give the title compound as apale yellow liquid (116.6 g, quantitative yield); 1H NMR (400 MHz,CDCl₃) δ 1.25 (t, 6H), 1.49-1.54 (2 x s, 9H), 2.78-2.83 (2 x s, 3H),3.50-3.66 (m, 4H), 4.42 (s, 2H), 5.49 (s, 1H), 7.22 (d, 2H) and 7.45 (d,2H) ppm.

Step 3: tert-ButylN-[[4-[hydroxyiminomethyl]phenyl]methyl]-N-methyl-carbamate

A biphasic solution of tert-butylN-[[4-(diethoxymethyl)phenyl]methyl]-N-methyl-carbamate (50.0 g, 154.6mmol) in 2-MeTHF (400.0 mL) and Na₂SO₄ (100.0 mL of 10% w/v, 70.40 mmol)was stirred at 8-10° C. in a 1-L, glass jacketed reactor. Hydroxylaminehydrochloride (46.38 mL of 5.0 M, 231.9 mmol) was added and the biphasicsolution was stirred at 30° C. for 16 hours. The reaction was dilutedwith MTBE (200.0 mL) and the layers separated. The organic phase waswashed with water (200.0 mL), dried (Na₂SO₄), filtered and concentratedin vacuo. The residue was diluted with heptane (200.0 mL) and theresultant suspension was stirred at ambient temperature for 30 minutes.The solid was collected by filtration to give the title compound as awhite solid (36.5 g, 89% Yield); 1H NMR (400 MHz, CDCl₃) δ 1.50 (s, 9H),2.88 (br s, 3H), 4.60 (s, 2H), 7.26 (d, 2H), 7.52 (d, 2H) and 8.15 (s,1H) ppm.

Step 4: tert-ButylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-N-methyl-carbamate

A suspension of tert-butylN-[[4-[hydroxyiminomethyl]phenyl]methyl]-N-methyl-carbamate (100 g,378.3 mmol) in isopropyl acetate (1.000 L) was stirred at ambienttemperature. N-Chlorosuccinimide (53.04 g, 397.2 mmol) was added andstirred at ambient temperature for 16 hours. The reaction waspartitioned with water (500.0 mL) and the phases separated. The organicphase was washed with water (500.0 mL) (2×), dried (Na₂SO₄), filteredand concentrated in vacuo to remove most of the solvent. Heptane (1.000L) was added and the mixture concentrated in vacuo to remove most of thesolvent. Heptane (1.000 L) was added and the resultant precipitateisolated by filtration. The filter-cake was washed with heptane (500 mL)and air-dried to give the title compound as an off-white powder (105.45g, 93% Yield); 1H NMR (400 MHz, CDCl₃) δ 1.48 (2 x s, 9H), 2.90 (2 x s,3H), 4.47 (s, 2H), 7.26 (d, 2H), 7.77 (d, 2H) and 8.82 (s, 1H) ppm.

Step 5: tert-ButylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-bromo-pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-methyl-carbamate

A suspension of tert-butylN-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-N-methyl-carbamate(100.0 g, 334.7 mmol) and tert-butylN-tert-butoxycarbonyl-N-[3-ethynyl-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]carbamate(121.2 g, 304.3 mmol) in DCM (1.212 L) was stirred at ambienttemperature. Triethylamine (33.87 g, 46.65 mL, 334.7 mmol) was added inone portion and the reaction stirred at ambient temperature for 16hours. The reaction was partitioned with water (606.0 mL) and the phasesseparated. The organic phase was washed with water (606.0 mL), dried(Na₂SO₄), filtered and concentrated in vacuo to near dryness. Heptane(363.6 mL) was added and the mixture concentrated to about 300 mL.Further heptane (1.212 L) was added and the mixture heated to 90° C.with stirring. The mixture was slowly cooled to ambient temperature andstirred at this temperature for 1 hour. The resultant precipitate wasisolated by filtration and the filter-cake washed with heptane (2×363.6mL) and air-dried to give the title compound as a beige solid (181.8 g,90% Yield); 1H NMR (400 MHz, CDCl₃) δ 1.41 (s, 18H), 1.51 (s, 9H), 2.88(2 x s, 3H), 4.50 (s, 2H), 7.36-7.38 (m, 3H), 7.86 (d, 2H) and 8.65 (s,1H) ppm.

Step 6:1-Bromo-4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfanyl-benzene

Sodium hydride (246.5 mg, 6.163 mmol) was added portionwise to a stirredsolution of 4-bromobenzenethiol (compound xxxi) (970.9 mg, 5.135 mmol)in DMF (10 mL) at 0° C. After stirring at this temperature for 15minutes 1,1,1,2,3,3,3-heptadeuterio-2-iodo-propane (1 g, 5.649 mmol) wasadded and the reaction allowed to warm to ambient temperature over 18hours. The reaction was quenched by the addition of water and themixture stirred for 10 minutes. The mixture was extracted with diethylether (×3) and the combined organic extracts washed with water (×2),brine (×2), dried (MgSO₄), filtered and concentrated in vacuo to givethe sub-title compound that was used directly without furtherpurification assuming 100% Yield and purity; 1H NMR (500 MHz, DMSO) δ7.25-7.37 (m, 2H) and 7.48-7.55 (m, 2H) ppm.

Step 7:1-Bromo-4-[1,2,2,2-tetradeuterio-1-(trideuteriomethl)ethyl]sulfonyl-benzene

mCPBA (2.875 g, 12.83 mmol) was added in portions to a stirred solutionof1-bromo-4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfanyl-benzene(1.223 g, 5.134 mmol) in DCM (20 mL) at 0° C. and the reaction allowedto warm to ambient temperature over 17 hours. The mixture was washed 1Maqueous NaOH (×2), saturated aqueous Na₂S₂O₃ (×3), brine (×1), dried(MgSO₄), filtered and concentrated in vacuo. The residue was purified bycolumn chromatography (ISCO Companion, 80 g column, eluting with 0 to40% EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title compoundas a colourless oil (1.19 g, 86% Yield); 1H NMR (500 MHz, DMSO) δ7.77-7.81 (m, 2H) and 7.88-7.92 (m, 2H) ppm.

Step 8:4,4,5,5-Tetramethyl-2-[4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonylphenyl]-1,3,2-dioxaborolane

Pd(dppf)Cl₂.DCM (179.8 mg, 0.2202 mmol) was added to a stirredsuspension of1-bromo-4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonyl-benzene(1.19 g, 4.404 mmol), bis(dipinacolato)diboron (1.342 g, 5.285 mmol) andKOAc (1.296 g, 13.21 mmol) in dioxane (10 mL). The reaction placed underan atmosphere of nitrogen via 5× nitrogen/vacuum cycles and the mixturewas heated at 80° C. for 4.5 hours. The reaction was cooled to ambienttemperature and the solvent removed in vacuo. The residue waspartitioned between Et₂O and water and the layers separated. The organiclayer was dried (MgSO₄), filtered and concentrated in vacuo. The residuewas dissolved in 30% EtOAc/Petroleum ether (35 mL) and 1.2 g of Florosilwas added. The mixture was stirred for 30 minutes then filtered, washingthe solids with further alliquots of 30% EtOAc/Petrol (×3). The filtratewas concentrated in vacuo and tritruated from 10% EtOAc/petroleum ether.The resultant solid was isolated by filtration, washed with petroleumether and dried in vacuo to give the sub-title compound as an off-whitesolid (1052.1 mg, 75% Yield); 1H NMR (400 MHz, DMSO) δ 1.33 (s, 12H),7.87 (d, J=8.4 Hz, 2H) and 7.94 (d, J=8.4 Hz, 2H) ppm.

Step 9: tert-ButylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonylphenyl]pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-methyl-carbamate

[1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (106.8mg, 0.1639 mmol) was added to a mixture of4,4,5,5-tetramethyl-2-[4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonylphenyl]-1,3,2-dioxaborolane(1.3 g, 4.098 mmol), tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-bromo-pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-methyl-carbamate(2.707 g, 4.098 mmol) and K₂CO₃ (1.133 g, 8.200 mmol) in toluene (9.100mL), EtOH (2.600 mL) and water (2.600 mL) and the reaction mixture wasdegassed with a flow of nitrogen (5 cycles).

The mixture was heated at 75° C. for 1.5 hours. The reaction was ccoledto ambient temperature and water (5.2 mL) was added. After stirring thelayers were separated and the organic layer dried (Na₂SO₄), filtered,and concentrated in vacuo. The residue was triturated with IPA and theresultant precipitate isolated by filtration, washed with IPA (3×4 mL)and dried in vacuo at 50° C. to give the title compound as a white solid(2.4 g, 76% Yield); 1H NMR (400 MHz, CDCl₃) δ 1.41 (s, 18H), 1.50 (s,9H), 2.85-2.89 (m, 3H), 4.50 (s, 2H), 7.36-7.38 (m, 3H), 7.87 (d, 2H),8.09 (d, 2H), 8.35 (d, 2H) and 9.06 (s, 1H) ppm.

Step 10:3-[3-[4-(Methylaminomethyl)phenyl]isoxazol-5-yl]-5-[4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonylphenyl]pyrazin-2-amine(compound II-4)

Concentrated HCl (3.375 g, 2.812 mL of 37% w/w, 34.25 mmol) was added toa solution of tert-butylN-[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]-6-[4-[1,2,2,2-tetradeuterio-1-(trideuteriomethyl)ethyl]sulfonylphenyl]pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-methyl-carbamate(2.2 g, 2.854 mmol) in acetone (28.60 mL) and the reaction heated atreflux for 7 hours. The reaction was cooled to ambient temperature andthe resultant precipitate isolated by filtration, washed with acetone(2×4.5 mL) and dried in vacuo at 50° C. to give the di-HCl salt of thetitle compound as a yellow solid (1.42 g, 92% Yield); 1H NMR (400 MHz,DMSO) δ 2.58 (t, 3H), 4.21 (t, 2H), 5.67 (br s, 2H), 7.74 (d, 2H), 7.85(s, 1H), 7.94 (d, 2H), 8.10 (d, 2H), 8.38 (d, 2H), 8.96 (s, 1H) and 9.33(br s, 2H) ppm; MS (ES+) 471.8.

Example 6 Synthesis of5-(4-(tert-butylsulfonyl)phenyl)-3-(3-(4-((methylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine(Compound I-2)

Step 1: Preparation of Compound 4-ii

A suspension of tert-butyl 4-((hydroxyimino)methyl)benzyl(methyl)carbamate (Compound 4-i) (650 g, 2.46 mol) in isopropyl acetate(6.5 L) is stirred at ambient temperature. N-Chlorosuccinimide (361 g,2.71 mol) is added and the reaction temperature maintained overnight at20-28° C. to ensure complete reaction. The reaction mixture is dilutedwith water (3.25 L) and EtOAc (1.3 L) and the phases are separated. Theorganic phase is washed with water (2×3.25 L), dried (Na₂SO₄), andconcentrated to a wet-cake. The concentrate is diluted with heptane (9.1L), ˜2 L of solvent removed, and then stirred at ambient temperature for2-20 h. The solid is collected by filtration. The filter-cake is washedwith heptane (2×975 mL) and dried to afford Compound 4-ii (692 g; 94%yield, 99.2 area % purity by HPLC) as a colorless powder.

Step 2: Preparation of tert-butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(methyl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(Compound 4-iv)

A suspension of tert-butylN-(5-bromo-3-ethynyipyrazin-2-yl)-N-tert-butoxycarbonyicarbamate(Compound 4-111)(1.59 kg, 3.99 mol) and tert-butyl4-(chloro(hydroxyimino)methyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate(1.31 kg, 4.39 mol; 1.10 equiv.) in CH₂Cl₂ (12.7 L) is stirred atambient temperature. Triethylamine (444 g, 611 mL, 4.39 mol) is added tothe suspension and the reaction temperature is maintained between 20-30°C. for 20-48 h to ensure complete reaction. The reaction mixture isdiluted with water (8 L) and thoroughly mixed, then the phases areseparated. The organic phase is washed with water (8 L), dried (Na₂SO₄),and then concentrated until about 1 L of CH₂Cl₂ remains. The concentrateis diluted with heptane (3.2 L) and re-concentrated at 40° C./200 torruntil no distillate is observed. The concentrate is stirred and furtherdiluted with heptane (12.7 L) to precipitate a solid. The suspension isstirred overnight. The solid is collected by filtration, washed withheptane (2×3 L) then dried to afford Compound 4-iv (2.42 kg; 92% yield,100 area % purity by HPLC) as a light tan powder. ¹H NMR (400 MHz,CDCl₃) δ 8.61 (s, 1H), 7.82 (d, J=8.2 Hz, 2H), 7.31 (m, 3H), 4.46 (br s,2H), 2.84 (br d, 3H), 1.57 (s, 2H), 1.44 (br s, 9H), 1.36 (s, 18H).

Step 3: Preparation of Compound 5-i

A mixture of tert-butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(methyl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(Compound 4-iv)(1.00 kg, 1.51 mol), K₂CO₃ (419 g, 3.02 mol), and(4-(isopropylsulfonyl)phenyl)boronic acid (345 g, 1.51 mol) in toluene(7.0 L) and water (2.0 L) was stirred and degassed with N₂ for 30 min.1,1′-bis(di-t-butylphosphino)ferrocen-dichloro-palladium(II)[Pd(dtbpf)Cl₂; 19.7 g, 30.3 mmol] was then added and degassed anadditional 20 min. The reaction mixture was warmed at 70° C. for atleast 1 h to ensure complete reaction. The reaction mixture was cooledto ambient temperature then filtered through Celite. The reaction vesseland filter pad are rinsed with toluene (2×700 mL). The filtrates arecombined and the phases are separated. The organic phase is stirred withBiotage MP-TMT resin (170 g) for 4-20 h. The resin is removed byfiltration through Celite and the filter pad is washed with toluene(2×700 mL). The filtrate and washings are combined and concentrated tonear dryness then diluted with i-PrOH (5.75 L) and re-concentrated. Theconcentrate is again dissolved in warm (45° C.) i-PrOH (5.75 L) and thencooled to ambient temperature with stirring to induce crystallizationthen stirred for around 16-20 h. The solid is collected by filtration,washed with i-PrOH (2×1 L), and dried to afford VRT-1018729 (967 g; 84%)as a beige powder. ¹H NMR (400 MHz, CDCl₃) δ 9.04 (s, 1H), 8.33 (d,J=8.6 Hz, 2H), 8.06 (d, J=8.5 Hz, 2H), 7.85 (d, J=8.1 Hz, 2H), 7.34 (m,3H), 4.47 (br s, 2H), 3.25 (hept, J=7.0 Hz, 1H), 2.85 (br d, 3H), 1.47(s, 9H), 1.38 (s, 18H), 1.33 (d, J=6.9 Hz, 6H).

Step 4: Preparation of Compound I-2.2HCl

A solution of Compound 5-i (950 g, 1.24 mol) in acetone (12.35 L) iswarmed to 40° C. then concentrated HCl (1.23 kg, 1.02 L of 37% w/w, 12.4mol) is added at a rate to maintain the reaction temperature between40-45° C. for at least 5 h to ensure complete reaction. The suspensionis cooled to below 30° C. and the solid collected by filtration. Thefilter-cake is washed with acetone (2×950.0 mL) then dried to affordCompound I-2.2HCl (578 g; 87% yield, 99.5 area % purity by HPLC) as ayellow powder. ¹H NMR (400 MHz, DMSO) δ 9.53 (br d, J=4.8 Hz, 2H), 8.93(s, 1H), 8.37 (d, J=8.5 Hz, 2H), 8.07 (d, J=8.3 Hz, 2H), 7.92 (d, J=8.6Hz, 2H), 7.84 (s, 1H), 7.75 (d, J=8.3 Hz, 2H), 4.23-4.15 (m, 2H), 3.43(hept, J=6.8 Hz, 1H), 2.55 (t, J=5.3 Hz, 3H), 1.17 (d, J=6.8 Hz, 6H).

Step 5: Preparation of Compound I-2.HCl from Compound I-2.2HCl

Two-Pot Process

A stirred suspension of Compound I-2.2HCl (874 g, 1.63 mol) in i-PrOH(3.50 L) and water (0.87 L) is warmed at 50° C. for 1-2 h, cooled toambient temperature, and stirred for 1-20 h. XRPD is performed on asmall sample to ensure that Compound I-2. 2HCl has been converted toanother form. The suspension is cooled to 5° C. and stirred for 1 h. Thesolid is collected by filtration then the filter-cake is washed with80/20 i-PrOH/water (2×874 mL), and briefly dried.

If XRPD shows the Compound I-2.HCl/anhydrate form, the solid is dried toafford Compound I-2.HCl/anhydrate (836 g, 99% yield, 99.2 area % purityby HPLC) as a yellow solid. ¹H NMR (400 MHz, DMSO) δ 9.38 (s, 2H), 8.96(s, 1H), 8.46-8.34 (m, 2H), 8.10 (d, J=8.3 Hz, 2H), 7.94 (d, J=8.6 Hz,2H), 7.85 (s, 1H), 7.75 (d, J=8.3 Hz, 2H), 7.23 (br s, 2H), 4.21 (s,2H), 3.47 (hept, J=6.7 Hz, 1H), 2.58 (s, 3H), 1.19 (d, J=6.8 Hz, 6H).

If XRPD shows the Compound I-2.HCl/hydrate form the solid is stirred infresh i-PrOH (3.50 L) and water (0.87 L) at 50° C. for at least 2 huntil XRPD shows complete conversion to Compound I-2.HCl/anhydrate. Thesuspension is then cooled to 5° C. and stirred for 1 h. The solid iscollected by filtration then the filter-cake is washed with 80/20i-PrOH/water (2×874 mL) then dried to afford Compound I-2.HCl/anhydrate.

Alternative Procedure (Single Pot) Used

Compound I-2.2HCl (392 g) is charged to the reactor. 4:1 IPA/water (8 L)is charged to a reactor and stirred at ambient temperature overnight.XRPD is used to confirm the conversion to the mono-HCl salt mono-hydrateform. The mixture is heated to 50° C. Seeds of CompoundI-2.HCl/anhydrate (16 g) are added and the mixture heated at 50° C.until XRPD confirms complete conversion to the desired anhydrate form.The mixture is to cooled to ambient, filtered and the solid washed with4:1 IPA/water (2×800 mL) then dried to afford Compound I-2.HCl/anhydrate(343 g, 94% yield).

Step 4: Alternate Method 1: Preparation of Compound I-2 Free Base

A solution of Compound 5-i (100 g, 131 mmol) in DCM (200 mL) was stirredat ambient temperature then TFA (299 g, 202 mL, 2.62 mol) was added.After 2 h reaction solution was cooled to 5° C. The reaction mixture wasdiluted with EtOH (1.00 L) over about 5 min resulting in a bright yellowsuspension. The suspension was cooled to 10° C. then NaOH (1.64 L of 2.0M, 3.28 mol) was added over 30 min then stirred at ambient temperatureovernight. The solid was collected by filtration then washed with water(2×400 mL), EtOH (2×200 mL) then dried to afford Compound I-2 free-base(57.0 g, 94% yield, 99.7 area % purity by HPLC) as a fine, yellowpowder. ¹H NMR (400 MHz, DMSO) δ 8.95 (s, 1H), 8.39 (d, J=8.5 Hz, 2H),7.95 (dd, J=11.6, 8.4 Hz, 4H), 7.78 (s, 1H), 7.51 (d, J=8.2 Hz, 2H),7.21 (br s, 2H), 3.72 (s, 2H), 3.47 (hept, J=6.8 Hz, 1H), 2.29 (s, 3H),1.19 (d, J=6.8 Hz, 6H).

Step 4: Alternate Method 2: Preparation of Compound I-2.HCl

A suspension of Compound I-2 free base (10.0 g, 21.6 mmol) in acetone(80 mL) was stirred and heated to 35° C. An aqueous solution of HCl(11.9 mL of 2.0 M, 23.8 mmol) diluted with water (8.0 mL) was added andthe mixture heated at 50° C. for 4 h. The suspension was allowed to coolto ambient temperature then stirred overnight. The solid was collectedby filtration. The filter-cake was washed with acetone (2×20 mL) thendried to afford 10.2 g Compound I-2 hydrochloride (95% yield) as ayellow powder.

Example 7 Synthesis of5-(4-(Isopropylsulfonyl)phenyl)-3-(3-(4-(tetrahydropyran-4-ylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (Compound I-3) Scheme: Example Synthesisof Compound I-3

Step 1: Preparation ofN-(4-(diethoxymethyl)benzyl)tetrahydro-2H-pyran-4-amine (A-2)

A solution of tetrahydro-2H-pyran-4-amine hydrochloride (1.13 kg, 8.21mol) in MeOH (14.3 L) is stirred at about 20° C. then Et₃N (1.06 kg,1.43 L, 8.21 mol) is added. The mixture is stirred for at least 5 minthen terephthalaldehyde diethyl acetal (1.43 kg, 6.84 mol) is addedwhile maintaining the reaction temperature between 20-25° C. The mixtureis stirred for at least 45 min to form the imine. NaBH₄ caplets (414 g,11.0 mol) are added while maintaining the reaction temperature belowabout 25° C. The mixture is stirred for 1 h after the addition iscompleted. The reaction mixture is quenched by adding 1 M NaOH (13.7 L)then extracted with MTBE. The organic solution was washed with brine(7.13 L) then dried (Na₂SO₄) and concentrated to afford Compound A-2(2197 g; 109% yield, 94.4 area % purity by HPLC) as a hazy oil. ¹H NMR(400 MHz, CDCl₃) δ 7.43 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.1 Hz, 2H), 5.49(s, 1H), 4.66 (br s, 1H), 4.03-3.91 (m, 2H), 3.82 (s, 2H), 3.69-3.47 (m,4H), 3.38 (td, J=11.6, 2.1 Hz, 2H), 2.78-2.65 (m, 1H), 1.90-1.81 (m,2H), 1.53-1.37 (m, 2H), 1.23 (t, J=7.1 Hz, 6H).

Step 2: Preparation of tert-butyl4-(diethoxymethyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate (A-3)

A mixture of N-(4-(diethoxymethyl)benzyl)tetrahydro-2H-pyran-4-amine(A-2) (2195 g, 7.48 mol) in CH₂Cl₂ (22.0 L) is stirred at 25° C. thendi-t-butyl dicarbonate (1.71 kg, 7.86 mol) is added. Et₃N (795 g, 1.10L) is then added while maintaining the reaction temperature between20-25° C. The reaction mixture is stirred at about 25° C. for 12-20 h.After the reaction is completed, the mixture is cooled to about 20° C.and quenched with 0.5 M aqueous citric acid (7.48 L, 3.74 mol) whilemaintaining the reaction temperature between 20-25° C. The organic phaseis collected, washed with sat. NaHCO₃ (6.51 L, 7.48 mol), washed withbrine (6.59 L), and dried (Na₂SO₄) then concentrated to affordtert-butyl 4-(diethoxymethyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate(A-3) (2801 g; 95% yield, 98.8 area % purity by HPLC) as a thick, amberoil. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J=8.1 Hz, 2H), 7.21 (d, J=7.9Hz, 2H), 5.49 (s, 1H), 4.39 (br s, 3H), 3.93 (br dd, J=10.8, 3.8 Hz,2H), 3.67-3.47 (m, 4H), 3.40 (br m, 2H), 1.68-1.59 (m, 4H), 1.39 (br s,9H), 1.23 (t, J=7.1 Hz, 6H).

Step 3: Preparation of tert-butyl4-((hydroxyimino)methyl)benzyhtetrahydro-2H-pyran-4-yl)carbamate (A-4)

A solution of tert-butyl4-(diethoxymethyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate (A-3) (2.80kg, 7.12 mol) in THF (28.0 L) and water (2.80 L) is stirred at about 20°C. Hydroxylamine hydrochloride (593 g, 8.54 mol) is added whilemaintaining the reaction temperature between 20-25° C. The reactionmixture is stirred at about 20° C. for 16 −20 h then diluted with CH₂Cl₂(8.4 L) and 50% brine (11.2 L) and stirred for at least 5 min. Thephases are separated then the organic phase is washed with 50% brine(2×2.8 L), dried (Na₂SO₄) and concentrated. The concentrate is dilutedwith MeOH (1.4 L) and re-concentrated. The concentrate is diluted withMeOH (14.0 L) and transferred to a reaction vessel. The solution iswarmed to about 25° C. then water (14.0 L) is added over about 1-1.5 h;after about 10 L of water is added, the mixture is seeded and a hazysuspension is observed. Additional water (8.4 L) is added over 1.5 h tofurther precipitate the product. After aging, the solid is collected byfiltration. The filter-cake is washed with heptane (5.6 L) and dried toafford tert-butyl4-((hydroxyimino)methyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate (A-4)(1678 g; 71%, 91.5 area % purity by HPLC) as an off-white powder. ¹H NMR(400 MHz, CDCl₃) δ 8.12 (s, 1H), 7.51 (d, J=8.2 Hz, 2H), 7.24 (d, J=7.9Hz, 2H), 4.40 (br s, 3H), 3.96 (dd, J=10.4, 3.6 Hz, 2H), 3.41 (br m,2H), 1.69-1.61 (m, 4H), 1.39 (br s, 9H).

Step 4: Preparation of (tert-butyl 4-(chloro(hydroxyimino)methyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate (A-4-i)

A suspension of (E)-tert-butyl4-((hydroxyimino)methyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate (A-4)(1662 g, 4.97 mol) in i-PrOAc (16.6 L) is stirred at 20° C. in areactor. N-chlorosuccinimide (730 g, 5.47 mol) is added maintainingabout 20° C. The suspension is stirred at about 20° C. to complete thereaction. The suspension is diluted with water (8.3 L) and stirred todissolve the solid. The phases are separated and the organic phase iswashed with water (8.3 L). The organic phase is concentrated thendiluted with i-PrOAc (831 mL). Heptane (13.3 L; 8 V) is slowly added toinduce crystallization. The thick suspension is then stirred for 1 h.The solid is collected by filtration; the filter-cake is washed withheptane (2×1.6 L; 2×1 V) and dried to afford (Z)-tert-butyl4-(chloro(hydroxyimino)methyl)benzyl (tetrahydro-2H-pyran-4-yl)carbamate(A-4-i) (1628 g; 89%, 98.0 area % purity by HPLC) as a white powder.

Step 5: Preparation of tert-butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(A-5)

A solution of tert-butyl4-(chloro(hydroxyimino)methyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate(A-4-i) (1.60 kg, 4.34 mol) and tert-butylN-(5-bromo-3-ethynyipyrazin-2-yl)-7V-tert-butoxycarbonylcarbamate(Compound A-4-ii) (1.73 kg, 4.34 mol) in CH₂Cl₂ (12.8 L) is stirred at20° C. Et₃N (483 g, 665 mL; 4.77 mol) is added and the reactiontemperature maintained below 30° C. The suspension stirred at 20° C. tocomplete the reaction then diluted with water (8.0 L) and agitated. Thephases are separated and the organic phase is washed with water (8.0 L)and then concentrated. i-PrOAc (1.6 L) is added and the mixture andheated at 50° C. Heptane (4.0 L) was slowly added then the suspension isallowed to cool to ambient temperature and stirred overnight. Additionalheptane (7.2 L) is added to the suspension and it is stirred for 1 h.The solid is collected by filtration. The filter-cake is washed withheptane (2×1.6 L) and dried to afford tert-butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(A-5) (2.478 kg; 78%, 97.8 area % purity by HPLC) as a fine, tan powder.¹H NMR (400 MHz, CDCl₃) δ 8.60 (s, 1H), 7.78 (d, J=8.3 Hz, 2H), 7.31 (m,3H), 4.42 (br m, 3H), 4.03-3.82 (m, 2H), 3.38 (br s, 2H), 1.60 (m, 4H),1.36 (s, 27H).

Step 6: Preparation of tert-butyltert-butoxycarbonyl(3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)-5-(4-(isopropyisulfonyl)phenyl)pyrazin-2-yl)carbamate

A mixture of tert-butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(A-5) (425 g, 582 mmol), K₂CO₃ (161 g, 1.16 mol; 2.0 equiv.), and(4-(isopropylsulfonyl)phenyl)boronic acid (133 g, 582 mmol) in toluene(2.98 L) and water (850 mL) is stirred and degassed with N₂ at ambienttemperature. The catalyst[1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II),(Pd(dtbpf)Cl₂; 1.90 g, 2.91 mmol) is added and the mixture is degassedfor an additional 10 min. The mixture is heated at 70° C. until thereaction is complete. The mixture is cooled to 50° C., diluted withwater (850 mL) and filtered through a bed of Celite. The phases areseparated. The organic phase is concentrated then the residue is dilutedwith EtOH (1.70 L) and re-concentrated. With mixing at 40° C., theconcentrate is diluted with EtOH (1.70 L) to induce crystallization. Thesuspension is cooled to 20° C. and stirred for 4 h. The solid iscollected by filtration. The filter-cake is washed with EtOH (2×425 mL)and air-dried to afford tert-butyltert-butoxycarbonyl(3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate(A-6) as a beige powder. The solid is dissolved in THF (2.13 L) andslurried with Biotage MP-TMT resin (48 g) at ambient temperature. Theresin is removed by filtration and the filtrate concentrated to removemost of the THF. The concentrate is diluted with EtOH (970 mL) andre-concentrated to about half the original volume. The concentrate isdiluted again with EtOH (970 mL) and mixed for 1 h at 40° C. Thesuspension is cooled to ambient temperature and the solid is collectedby filtration then dried to afford tert-butyltert-butoxycarbonyl(3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate(A-6) (416 g; 86% yield, 99.3 area % purity by HPLC) as a white powder.¹H NMR (400 MHz, CDCl₃) δ 9.04 (s, 1H), 8.38-8.28 (m, 2H), 8.10-8.01 (m,2H), 7.82 (d, J=8.2 Hz, 2H), 7.34 (m, 3H), 4.44 (br s, 2H), 3.94 (dd,J=10.5, 3.5 Hz, 2H), 3.40 (br s, 2H), 3.25 (hept, J=6.8 Hz, 1H), 1.65(m, 4H), 1.38 (br s, 27H), 1.33 (d, J=6.9 Hz, 6H).

Step 7: Preparation of5-(4-(isopropyisulfonyl)phenyl)-3-(3-(4-(((tetrahydro-2H-pyran-4-y)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine(I-3) Freebase Form

A suspension of tert-butyltert-butoxycarbonyl(3-(3-(4-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate(A-6) (410 g; 492 mmol) in CH₂Cl₂ (410 mL) is stirred at ambienttemperature in a flask. TFA (841 g, 568 mL; 7.4 mol) is added whilemaintaining the reaction temperature between 20-25° C. The solution isstirred at ambient temperature for about 3 h when analysis showsreaction completion. The solution is cooled to about 5-10° C. anddiluted with EtOH (3.3 L) while maintaining the temperature below 20° C.A 5.0 M aqueous solution of NaOH (1.77 L; 8.85 mol) is added whileallowing the reaction temperature to rise from about 14° C. to about 42°C. The suspension is heated at 70-75° C. for 6 h while removingdistillate. The suspension is allowed to cool to ambient temperature.The solid is collected by filtration and the filter-cake is washed withwater (4×1.64 L). The filter-cake is washed with EtOH (2×820 mL) anddried to afford5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(((tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (Compound I-1) (257 g; 98% yield, 99.5area % purity by HPLC) as a yellow powder.

¹H NMR (400 MHz, DMSO) δ 8.94 (s, 1H), 8.44-8.33 (m, 2H), 7.94 (t, J=8.2Hz, 4H), 7.76 (s, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.20 (s, 2H), 3.83 (m,1H), 3.80 (s, 3H), 3.46 (hept, J=6.8 Hz, 1H), 3.25 (td, J=11.4, 2.1 Hz,2H), 2.66-2.54 (m, 1H), 1.79 (br dd, 2H), 1.36-1.22 (m, 2H), 1.19 (d,J=6.8 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 167.57, 151.76, 141.07, 137.58,135.75, 129.16, 128.53, 126.57, 126.41, 125.69, 124.52, 102.13, 65.83,54.22, 52.60, 49.19, 33.18, 15.20.

Compound Analytical Data

Cmpd LCMS LCMS No. ES + (Rt min) HNMR I-1 — — ¹H NMR (400 MHz, DMSO) δ9.63 (d, J = 4.7 Hz, 2H), 9.05 (s, 1H), 8.69 (d, J = 5.2 Hz, 1H), 8.21(s, 1H), 8.16-8.03 (m, 3H), 7.84 (t, J = 4.1 Hz, 3H), 7.34 (br s, 2H),4.40-4.18 (m, 2H), 3.94 (dd, J = 11.2, 3.9 Hz, 2H), 3.32 (t, J = 11.2Hz, 3H), 2.17-2.00 (m, 2H), 1.81 (s, 6H), 1.75 (dd, J = 12.1, 4.3 Hz,2H). I-2 — — ¹H NMR (400 MHz, DMSO) δ 8.94 (s, 1H), 8.44-8.33 (m, 2H),7.94 (t, J = 8.2 Hz, 4H), 7.76 (s, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.20(s, 2H), 3.83 (m, 1H), 3.80 (s, 3H), 3.46 (hept, J = 6.8 Hz, 1H), 3.25(td, J = 11.4, 2.1 Hz, 2H), 2.66-2.54 (m, 1H), 1.79 (br dd, 2H),1.36-1.22 (m, 2H), 1.19 (d, J = 6.8 Hz, 6H). II-1 466.2 0.83 1H NMR (500MHz, DMSO) 9.10 (d, J = 5.8 Hz, 2H), 8.97 (s, 1H), 8.42-8.37 (m, 2H),8.15-8.08 (m, 2H), 7.99-7.92 (m, 2H), 7.85 (s, 1H), 7.75-7.69 (m, 2H),7.22 (br s, 2H), 3.48 (hept, J = 6.8 Hz, 1H), 2.60 (t, J = 5.4 Hz, 3H),1.20 (d, J = 6.8 Hz, 6H). II-2 469.1 0.82 1H NMR (500 MHz, DMSO) 9.14(s, 2H), 8.96 (s, 1H), 8.42-8.36 (m, 2H), 8.13-8.08 (m, 2H), 7.99-7.91(m, 2H), 7.85 (s, 1H), 7.78-7.68 (m, 2H), 7.21 (s, 2H), 3.48 (dq, J =13.6, 6.7 Hz, 1H), 1.20 (d, J = 6.8 Hz, 6H). II-3 467.2 0.78 1H NMR (500MHz, DMSO) 9.11 (s, 2H), 8.97 (s, 1H), 8.39 (d, J = 8.7 Hz, 2H), 8.11(d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.7 Hz, 2H), 7.85 (s, 1H), 7.72 (d, J= 8.4 Hz, 2H), 7.23 (s, 2H), 4.25-4.19 (m, 2H), 3.47 (tt, J = 14.0, 6.9Hz, 1H), 1.20 (d, J = 6.8 Hz, 6H). II-4 471.8 0.83 1H NMR (400 MHz,DMSO) δ 2.58 (t, 3H), 4.21 (t, 2H), 5.67 (br s, 2H), 7.74 (d, 2H), 7.85(s, 1H), 7.94 (d, 2H), 8.10 (d, 2H), 8.38 (d, 2H), 8.96 (s, 1H) and 9.33(br s, 2H) ppm

Intermediates Example 8 Preparation of Oxime 5a

Step 1b

Add MeOH (28.00 L) and 4-(diethoxymethyl)benzaldehyde (Compound 1b)(3500 g, 16.81 mol) into a reactor at 20° C. Add methylamine, 33% inEtOH (1.898 kg, 2.511 L of 33% w/w, 20.17 mol) maintaining 20-30° C.then stir for 1.5 h to form the imine. Add NaBH₄ (381.7 g, 10.09 mol)caplets maintaining the temperature between 20-30° C. Stir at roomtemperature for at least 30 min to ensure complete reaction. Add aqueousNaOH (16.81 L of 2.0 M, 33.62 mol) maintaining approximately 20° C. AddMTBE (17.50 L) and brine (7.0 L), stir for at least 5 min then allow thephases to separate. Extract the aqueous layer with MTBE (7.0 L) thencombine the organic phases and wash with brine (3.5 L) then dry (Na₂SO₄)then concentrate to 6 L. The biphasic mixture was transferred to aseparatory funnel and the aqueous phase removed. The organic phase wasconcentrated to afford 1-(4-(diethoxymethyl)phenyl)-N-methylmethanamine(Compound 2b) (3755 g, 16.82 mol, 100% yield) as an oil. ¹H NMR (400MHz, CDCl₃) δ 7.43 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.1 Hz, 2H), 5.49 (s,1H), 3.75 (s, 2H), 3.68-3.46 (m, 4H), 2.45 (s, 3H), 1.23 (t, J=7.1 Hz,6H).

Steps 2b and 3b

Add 2-MeTHF (15.00 L) and1-(4-(diethoxymethyl)phenyl)-N-methylmethanamine (Compound 2b) (3750 g,16.79 mol) to a reactor at 20° C. Add a solution of Boc anhydride (3.848kg, 4.051 L, 17.63 mol) in 2-MeTHF (7.500 L) maintaining approximately25° C. Stir for at least 30 min to ensure complete conversion totert-butyl 4-(diethoxymethyl)benzyl(methyl)carbamate (Compound 3b), thenadd a solution of Na₂SO₄ (1.192 kg, 8.395 mol) in water (11.25 L). Heatto 35° C. then add a solution of hydroxylamine hydrochloride (1.750 kg,25.18 mol) in water (3.75 L) then stir for at least 6 h to ensurecomplete reaction. Cool to 20° C., stop the stirring and remove theaqueous phase. Wash the organic layer with brine (3.75 L), dry (Na₂SO₄),filter and concentrate to about 9 L. Add heptane (15.00 L) andcrystalline tert-butyl 4-((hydroxyimino)methyl)benzyl(methyl) carbamate(Compound 5a) (1.0 g portions every 10 min) until nucleation wasevident, then concentrate to afford a solid slurry. Add heptane (3.75 L)then cool to room temp and filter. Wash with heptane (5.625 L) then dryto afford tert-butyl 4-((hydroxyimino)methyl) benzyl(methyl)carbamate(Compound 5a) (4023 g, 15.22 mol, 91% yield, 97.2 area % purity by HPLC)as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.54 (d,J=8.1 Hz, 2H), 7.25 (br d, 2H), 4.44 (br s, 2H), 2.83 (br d, 3H), 1.47(br s, 9H).

The compound of formula A-4-ii may be made according to the stepsoutlined in Scheme C. Sonogashira coupling reactions are known in theart (see e.g., Chem. Rev. 2007, 874-922). In some embodiments, suitableSonogashira coupling conditions comprise adding 1 equivalent of thecompound of formula C-1, 1 equivalent of TMS-acetylene, 0.010equivalents of Pd(PPh₃)₂Cl₂, 0.015 equivalents of CuI and 1.20equivalents of NMM in isopropanol. The product can be isolated by addingwater to the alcoholic reaction mixture.

Amine salts of a product maybe formed by dissolving the amine in acommon organic solvent and adding an acid. Examples of suitable solventsinclude chlorinated solvents (e.g., dichloromethane (DCM),dichloroethane (DCE), CH₂Cl₂, and chloroform), ethers (e.g., THF,2-MeTHF and dioxane), esters (e.g., EtOAc, IPAC) and other aproticsolvents. Examples of suitable acids include but are not limited to HCl,H₃PO₄, H₂SO₄, MSA, and PTSA. In some embodiments, the solvent is IPACand the acid is PTSA. In some embodiments, the acid addition salt isconverted back to the free amine base in the presence of a suitablesolvent and a suitable base. Suitable solvents include EtOAc, IPAC,dichloromethane (DCM), dichloroethane (DCE), CH₂Cl₂, chloroform,2-MeTHF, and suitable bases include NaOH, NaHCO₃, Na₂CO₃, KOH,KHCO₃,K₂CO₃, and Cs₂CO₃. In some embodiments, the suitable solvent isEtOAc and the suitable base is KHCO₃.

The amine of Compound C-2 may be protected with various amine protectinggroups, such as Boc (tert-butoxycarbonyl). Introduction of Bocprotecting groups is known in the art (see e.g. Protecting Groups inOrganic Synthesis, Greene and Wuts). In some embodiments, suitableconditions involve adding 1.00 equivalents of the amine, 2.10equivalents of di-tert-butyl dicarbonate, and 0.03 equivalents of DMAPin EtOAc.

Reduction in Pd is achieved by treating with a metal scavenger (silicagel, functionalized resins, charcoal). In some embodiments, suitableconditions involve adding charcoal.

The TMS (trimethylsilyl) protecting group on Compound C-3 may be removedvia conditions known to one of skill in the art. In some embodiments,TMS removal conditions comprise reacting the TMS-protected compound witha suitable base in a suitable solvent. Examples of suitable solventsinclude chlorinated solvents (e.g., dichloromethane (DCM),dichloroethane (DCE), CH₂Cl₂, and chloroform), ethers (e.g., THF,2-MeTHF and dioxane), esters (e.g., EtOAc, IPAC), other aprotic solventsand alcohol solvents (e.g., MeOH, EtOH, iPrOH). Examples of suitablebases include but are not limited to (e.g., NaOH, KOH, K₂CO₃, Na₂CO₃).In certain embodiments, suitable conditions comprise adding 1.00equivalents of the TMS-protected acetylene, 1.10 equivalents of K₂CO₃,EtOAc and EtOH. In some embodiments, the alcoholic solvent, such asEtOH, is added last in the reaction. In some embodiments the productacetylene is isolated by adding water.

Example 9 Synthesis of Compound A-4-ii

Step 1: Preparation of5-bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-amine (Compound C-2)

Charge isopropanol (8.0 L) to a reactor the stir and sparge with astream of N₂. Add 3,5-dibromopyrazin-2-amine (Compound C-1) (2000 g,7.91 moles), Pd(PPh₃)₂Cl₂ (56 g, 0.079 moles), CuI (23 g, 0.119 moles),and NMM (1043 mL, 9.49 moles) to the reactor under a N₂ atmosphere.Adjust the reaction temperature to 25° C. Purge the reactor with N₂ bydoing at least three vacuum/N₂ purge cycles. Charge TMS-acetylene (1.12L, 7.91 moles) to the reaction mixture and maintain the reactiontemperature below 30° C. When the reaction is complete lower thetemperature of the reaction mixture to 15° C. then add water (10 L) andstir for at least 2 h. The solid is collected by filtration washing thesolid with 1:1 IPA/water (2×6 L). The filter cake is dried under vacuumthen charged to a reactor and dissolved in EtOAc (12.5 L). PTSA hydrate(1.28 kg, 6.72 mol) is charged as a solid to the reactor. The mixture isstirred at ambient temperature for at least 5 h then the solid iscollected by filtration, washed with 1:1 heptane/EtOAc (3.5 L) followedby heptane (3.5 L). The filter cake is dried to afford5-bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-amine(Compound C-2) as aPTSA salt (2356 g, 67% yield, 98.9 area % purity by HPLC). ¹H NMR (400MHz, DMSO) δ 8.12 (s, 1H), 7.48 (d, J=8.1 Hz, 2H), 7.12 (d, J=8.0 Hz,2H), 2.29 (s, 3H), 0.26 (s, 9H).

Steps 2 and 3

Step 2: Preparation of tert-butylN-tert-butoxycarbonyl-N-[5-bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-yl]carbamate(Compound C-3)

A solution of 5-bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-amine PTSAsalt (Compound C-2) (2350 g, 5.31 mol) in EtOAc (11.5 L) is stirred witha 20% w/w aq. solution of KHCO₃ (4.5 kg, 1.5 eq.) for at least 30 min.The layers are separated and the organic layer is concentrated thendissolved in EtOAc (7 L) and added to a reactor. DMAP (19.5 g, 0.16 mol)is added followed a solution of Boc₂O (2436 g, 11.16 mol) in EtOAc (3 L)is added lowly. The reaction is stirred for at least 30 min to ensurecomplete reaction then activated charcoal (Darco G-60, 720 g) and Celite(720 g) are added and stirred for at least 2 h. The mixture is filteredwashing the solid pad with EtOAc (2×1.8 L). The filtrate is concentratedto afford tert-butylN-tert-butoxycarbonyl-N-[5-bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-yl]carbamate (Compound C-3) that is used directly in the nextstep.

Step 3: Preparation of tert-butylN-5-bromo-3-ethynylpyrazin-2-yl)-N-tert-butoxyearbonylearbamate(Compound A-4-ii)

K₂CO₃ (811 g, 5.87 mol) is charged to a reactor followed by a solutionof Compound C-3 (2300 g, 4.89 mol) dissolved in EtOAc (4.6 L) agitationstarted. EtOH (9.2 L) is added slowly and the mixture stirred for atleast 1 h to ensure that the reaction is complete then water (4.6 L) isadded and stirred for at least 2 h. The solid is collected by filtrationand washed with 1:1 EtOH/water (4.6 L followed by 2.3 L) followed byEtOH (2.3 L). The filter cake is dried to afford tert-butylN-(5-bromo-3-ethynyipyrazin-2-yl)-N-tert-butoxycarbonylcarbarnate(Compound A-4-ii) (1568 g, 78% yield, 97.5 area % by HPLC). ¹H NMR (400MHz, CDCl₃) δ 8.54 (s, 1H), 3.52 (s, 1H), 1.42 (s, 18H).

Solid Forms of Compound I-2

Compound I-2 has been prepared in various solid forms, including saltsand co-solvates. The solid forms of the present invention are useful inthe manufacture of medicaments for the treatment of cancer. Oneembodiment provides use of a solid form described herein for treatingcancer. In some embodiments, the cancer is pancreatic cancer ornon-small cell lung cancer. Another embodiment provides a pharmaceuticalcomposition comprising a solid form described herein and apharmaceutically acceptable carrier.

Applicants describe herein five novel solid forms of Compound I-2. Thenames and stoichiometry for each of these solid forms are provided inTable S-1 below:

TABLE S-1 Example Forms Stoichiometry Example 13 Compound I-2 free baseN/A Example 14 Compound I-2•HCl 1:1 Example 15 Compound I-2•2HCl 1:2Example 16 Compound I-2•HCl•H₂O 1:2:1 Example 17 Compound I-2•HCl•2H₂O1:1:2

Solid state NMR spectra were acquired on the Bruker-Biospin 400 MHzAdvance III wide-bore spectrometer equipped with Bruker-Biospin 4 mm HFXprobe. Samples were packed into 4 mm ZrO₂ rotors (approximately 70 mg orless, depending on sample availability). Magic angle spinning (MAS)speed of typically 12.5 kHz was applied. The temperature of the probehead was set to 275K to minimize the effect of frictional heating duringspinning. The proton relaxation time was measured using ¹H MAS T₁saturation recovery relaxation experiment in order to set up properrecycle delay of the ¹³C cross-polarization (CP) MAS experiment. Therecycle delay of¹³C CPMAS experiment was adjusted to be at least 1.2times longer than the measured ¹H T₁ relaxation time in order tomaximize the carbon spectrum signal-to-noise ratio. The CP contact timeof ¹³C CPMAS experiment was set to 2 ms. A CP proton pulse with linearramp (from 50% to 100%) was employed. The Hartmann-Hahn match wasoptimized on external reference sample (glycine). Carbon spectra wereacquired with SPINAL 64 decoupling with the field strength ofapproximately 100 kHz. The chemical shift was referenced againstexternal standard of adamantane with its upheld resonance set to 29.5ppm.

XRPD data for Examples 13-14 were measured on Bruker D8 Advance System(Asset V014333) equipped with a sealed tube Cu source and a Vantec-1detector (Bruker AXS, Madison, Wis.) at room temperature. The X-raygenerator was operating at a voltage of 40 kV and a current of 40 mA.The powder sample was placed in a shallow silicon holder. The data wererecorded in a reflection scanning mode (locked coupled) over the rangeof 3°-40° 2 theta with a step size of 0.0144° and a dwell time of 0.25 s(105 s per step). Variable divergence slits were used.

Example 10 Compound I-2 (Free Base)

Compound I-2 free base can be formed according to the methods describedin Example 6, Step 4: Alternate Method 1.

XRPD of Compound I-2 (Free Base)

FIG. 1a shows the X-ray powder diffractogram of the sample which ischaracteristic of crystalline drug substance.

Representative XRPD peaks from Compound I-2 free base:

XRPD Angle Peaks (2-Theta ± 0.2) Intensity % 1 23.8 100.0 *2 14.2 43.9 322.5 39.3 *4 25.6 31.1 5 19.3 28.6 6 27.2 27.6 7 17.0 25.4 *8 18.1 25.29 17.6 19.6 10 20.2 17.2 11 28.3 15.6 12 20.8 14.5 13 29.9 14.5 14 33.214.3 15 30.1 13.5 16 26.8 13.4 *17 22.0 12.3 18 36.5 12.3 19 31.8 12.220 34.6 11.5 21 31.1 11.2 22 34.0 11.0 23 30.6 10.9 *24 11.1 10.6 2513.3 10.6

Thermo Analysis of Compound I-2 Free Base

A thermal gravimetric analysis of Compound I-2 free base was performedto determine the percent weight loss as a function of time. The samplewas heated from ambient temperature to 350° C. at the rate of 10° C./minon TA Instrument TGA Q5000 (Asset V014258). FIG. 2a shows the TGA resultwith a one-step weight loss before evaporation or thermal decomposition.From ambient temperature to 215° C., the weight was ˜1.9%.

Differential Scanning Calorimetry of Compound I-2 Free Base

The thermal properties of Compound I-2 free base were measured using theTA Instrument DSC Q2000 (Asset V014259). A Compound I-2 free base sample(1.6900 mg) was weighed in a pre-punched pinhole aluminum hermetic panand heated from ambient temperature to 350° C. at 10° C./min. Oneendothermic peak is observed at 210° C. with its onset temperature at201° C. (FIG. 3a ). The enthalpy associated with the endothermic peak is78 J/g.

Solid State NMR of Compound I-2 Free Base

-   13C CPMAS on Compound I-2 free base-   275K; 1H T1=1.30 s-   12.5 kHz spinning; ref adamantane 29.5 ppm-   For the full spectrum, see FIG. 4 a.

Representative Peaks

Chem Shift Intensity Peak # [ppm] [rel]  1* 171.0 28.7 2 163.7 21.4  3*152.1 26.3 4 143.1 57.3  5* 141.2 38.8 6 138.8 30.0 7 132.4 62.1 8 130.952.3 9 130.0 70.7 10* 126.6 100.0 11  123.5 34.3 12  101.3 34.1 13  57.684.3 14* 38.1 42.6 15* 19.2 48.8 16  18.1 53.3

Crystal Structure of Compound I-2 Free Base

The free form of Compound I-2 was prepared from the Compound I-2 HClsalt. 200 mg Compound I-2 HCl salt was added to 1 mL of 6N NaOHsolution. 20 mL of dichloromethane was used to extract the free form.The dichloromethane layer was dried over K₂CO₃. The solution wasfiltered off and 5 mL of n-heptane was added to it. Crystals wereobtained by slow evaporation of the solution at room temperature overnight.

Most crystals obtained were thin plates. Some prismatic shape crystalswere found among them.

A yellow prismatic crystal with dimensions of 0.2×0.1×0.1 mm³ wasselected, mounted on a MicroMount and centered on a Bruker APEX IIdiffractometer. Three batches of 40 frames separated in reciprocal spacewere obtained to provide an orientation matrix and initial cellparameters. Final cell parameters were obtained and refined after datacollection was completed based on the full data set.

A diffraction data set of reciprocal space was obtained to a resolutionof 116.96° 2θ angle using 0.5° steps with 10 s exposure for each frame.Data were collected at 100 (2) K temperature with a nitrogen flowcryosystem. Integration of intensities and refinement of cell parameterswere accomplished using APEXII software.

Crystal Data

C₂₄H₂₅N₅O₃S M_(r) = 463.55 Monoclinic, P2₁/n a = 8.9677 (1) Å b =10.1871 (1) Å c = 24.5914 (3) Å β = 100.213 (1)° V = 2210.95 (4) Å³ Z =4

Example 11 Compound I-2.HCl

Compound I-2.HCl can be formed according to the methods described inExample 6, Step 4: Alternate Method 2 and Example 6, Step 5.

XRPD of Compound I-2.HCl

FIG. 1b shows the X-ray powder diffractogram of the sample which ischaracteristic of crystalline drug substance.

Representative XRPD peaks from Compound I-2.HCl

XRPD Angle Peaks (2-Theta ± 0.2) Intensity % 1 14.4 100.0 *2 13.5 89.1 320.9 45.8 4 16.4 41.9 5 23.6 33.8 6 27.2 29.6 *7 28.8 26.0 8 16.8 23.3 927.6 21.3 *10 15.0 21.1 *11 18.8 20.8 12 30.5 13.3 *13 15.4 13.0 14 26.912.7

Thermo Analysis of Compound I-2.HCl

A thermal gravimetric analysis of Compound I-2.HCl was performed todetermine the percent weight loss as a function of time. The sample washeated from ambient temperature to 350° C. at the rate of 10° C./min onTA Instrument TGA Q5000 (Asset V014258). FIG. 2b shows the TGA resultwith a two-step weight loss before evaporation or thermal decomposition.From ambient temperature to 100° C., the weight was ˜1.1%, and from 110°C. to 240° C. the weight loss is ˜0.8%.

Differential Scanning Calorimetry of Compound I-2.HCl

The thermal properties of Compound I-2.HCl were measured using the TAInstrument DSC Q2000 (Asset V014259). A Compound I-2.HCl sample (3.8110mg) was weighed in a pre-punched pinhole aluminum hermetic pan andheated from ambient temperature to 350° C. at 10° C./min. Oneendothermic peak is observed at 293° C. with its onset temperature at291° C. (FIG. 3b ). The enthalpy associated with the endothermic peak is160.3 J/g. The second endothermic peak is around 321° C. Both peaks werecoupled with sample evaporation and decomposition.

Solid State NMR of Compound I-2.HCl

-   ¹⁵CPMAS on Compound I-2.HCl-   275K; 12.5 kHz spinning; ref adamantane 29.5 ppm-   For the full spectrum, see FIG. 4 b.

Representative Peaks

Chem Shift Intensity Peak # [ppm] [rel]  1* 171.7 47.42 2 161.9 28.72 3* 153.4 28.94 4 144.8 42.57 5 142.9 54.14 6 138.7 44.06 7 136.7 60.06 8* 132.9 100 9 131.2 72.62 10  129.8 73.58 11  127.9 63.71 12  125.479.5 13  124.1 34.91 14  100.7 53.52 15  54.5 62.56 16  53.9 61.47 17*31.8 61.15 18  17.0 74.78 19* 15.7 77.79

Crystal Structure of Compound I-2.HCl

180 mg Compound I-2.HCl was added to a vial with 0.8 mL 2-propanol and0.2 mL water. The sealed vial was kept in an oven at 70° C. for twoweeks. Diffraction quality crystals were observed.

A yellow needle shape crystal with dimensions of 0.15×0.02×0.02 mm³ wasselected, mounted on a MicroMount and centered on a Bruker APEX IIdiffractometer (V011510). Three batches of 40 frames separated inreciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were collected andrefined was completed based on the full data set.

A diffraction data set of reciprocal space was obtained to a resolutionof 106° 2θ angle using 0.5° steps with exposure times 20 s each framefor low angle frames and 60 s each frame for high angle frames. Datawere collected at room temperature.

To obtain the data in table 1, dry nitrogen was blown to the crystal at6 Litre/min speed to keep the ambient moisture out. Data in table 2 wasobtained without nitrogen. Integration of intensities and refinement ofcell parameters were conducted using the APEXII software. The wateroccupancy can vary between 0 and 1.

TABLE 1 C₂₄H₂₆ClN₅O₃S M_(r) = 500.01 Monoclinic, P2₁/n a = 5.3332 (2) Åb = 35.4901 (14) Å c = 13.5057 (5) Å β = 100.818 (2)° V = 2510.87 (17)Å³

TABLE 2 C₂₄H₂₈ClN₅O₄S Mr = 518.02 Monoclinic, P21/n a = 5.4324 (5) Å b =35.483 (4) Å c = 13.3478 (12) Å β = 100.812 (5)° V = 2527.2 (4) Å3

CHN Elemental Analysis

CHN elemental analysis of Compound I-2.HCl suggest a mono HCl salt.

Element C H N Cl % Theory 57.60 5.20 14.00 7.10 C₂₄H₂₅N₅O3S•HCl % Found56.52 5.38 13.69 7.18

Example 12 Compound I-2.2HCl

Compound I-2.2HCl can be formed according to the methods described inExample 6, Step 4.

XRPD of Compound I-2.2HCl

The XRPD patterns are acquired at room temperature in reflection modeusing a Bruker D8 Discover system (Asset Tag V012842) equipped with asealed tube source and a Hi-Star area detector (Bruker AXS, Madison,Wis.). The X-Ray generator is operated at a voltage of 40 kV and acurrent of 35 mA. The powder sample is placed in a nickel holder. Twoframes are registered with an exposure time of 120 s each. The data issubsequently integrated over the range of 4.5°-39° 2 theta with a stepsize of 0.02° and merged into one continuous pattern.

FIG. 1c shows the X-ray powder diffractogram of the sample which ischaracteristic of crystalline drug substance.

Representative XRPD peaks from Compound I-2.2HCl

XRPD Angle Peaks (2-Theta ± 0.2) Intensity % 1 16.8 100.0 *2 15.1 92.2 326.2 91.3 4 27.5 91.3 5 27.2 91.2 6 11.8 89.0 7 29.8 89.0 8 22.8 88.8 915.7 88.1 *10 18.5 87.5 11 8.4 87.4 12 12.8 86.6 *13 11.5 86.0 14 14.686.0 15 20.1 86.0 *16 13.1 85.9 17 16.0 85.9 18 17.3 85.7 19 23.9 85.720 19.2 85.4 *21 5.7 85.2 22 21.3 85.2 23 25.3 84.9 24 28.7 84.7 25 24.384.1

Thermo Analysis of Compound I-2.2HCl

A thermal gravimetric analysis of Compound I-2.2HCl was performed on theTA Instruments TGA model Q5000. Compound I-2.2HCl was placed in aplatinum sample pan and heated at 10° C./min to 350° C. from roomtemperature. FIG. 2c shows the TGA result, which demonstrates a weightloss of 7.0% from room temperature to 188° C., which is consistent withthe loss of 1 equivalent of HCl (6.8%). The onset temperature ofdegradation/melting is 263° C.

Differential Scanning Calorimetry of Compound I-2.2HCl

A DSC thermogram for Compound I-2.2HCl drug substance lot 3 was obtainedusing TA Instruments DSC Q2000. Compound I-2.2HCl was heated at 2°C./min to 275° C. from −20° C., and modulated at ±1° C. every 60 sec.The DSC thermogram (FIG. 3c ) reveals an endothermic peak below 200° C.,which could corresponds to the loss of 1 equivalent of HCl.Melting/recrystallization occurs between 215-245° C., followed bydegradation.

Solid State NMR of Compound I-2.2HCl

-   ¹³C CPMAS on Compound I-2.2HCl-   275K; 1H T1=1.7 s-   12.5 kHz spinning; ref adamantane 29.5 ppm-   For the full spectrum, see FIG. 4c .

Peak # Chem Shift [ppm] Intensity [rel]  1* 166.5 32.6 2 160.7 24.7 3145.3 15.0  4* 137.6 56.0  5* 136.1 100.0 6 134.2 22.7 7 132.4 55.9 8130.0 54.9 9 127.7 70.7 10  125.9 97.1 11  124.7 59.0 12  123.8 91.4 13 123.2 56.0 14  101.6 37.7 15  56.1 60.3 16  50.7 45.6 17* 34.2 56.8 18 18.4 63.5 19* 16.4 70.32

Crystal Structure of Compound I-2.2HCl

180 mg Compound I-2.HCl was added to a vial with 0.8 mL 2-propanol and0.2 mL water. The sealed vial was kept in an oven at 70° C. for twoweeks. Diffraction quality crystals were observed.

A yellow needle shape crystal with dimensions of 0.15×0.02×0.02 mm³ wasselected, mounted on a MicroMount and centered on a Bruker APEX IIdiffractometer (V011510). Three batches of 40 frames separated inreciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were collected andrefined was completed based on the full data set.

A diffraction data set of reciprocal space was obtained to a resolutionof 106° 2θ angle using 0.5° steps with exposure times 20 s each framefor low angle frames and 60 s each frame for high angle frames. Datawere collected at room temperature. Dry nitrogen was blown to thecrystal at 6 Litre/min speed to keep the ambient moisture out.Integration of intensities and refinement of cell parameters wereconducted using the APEXII software.

Crystal Data

C₂₄H₂₆ClN₅O₃S M_(r) = 500.01 Monoclinic, P2₁/n a = 5.3332 (2) Å b =35.4901 (14) Å c = 13.5057 (5) Å β = 100.818 (2)° V = 2510.87 (17) Å³

Example 13 Compound I-2.HCl.H₂O

Compound I-2.HCl.H₂O can be formed from Compound I-2.2 HCl. (E29244-17)A suspension of Compound I-2.2 HCl (10.0 g, 18.6 mmol) in isopropylalcohol (40 mL) and water (10 mL) is warmed at 50° C. for about 1 h andthen cooled to below 10° C. The solid is collected by filtration. Thefilter-cake is washed with 80/20 isopropyl alcohol/water (2×10 mL) andair-dried to afford Compound I-2.HCl.2H₂O as a yellow powder.

XRPD of Compound I-2.HCl.H₂O

The XRPD patterns are acquired at room temperature in reflection modeusing a Bruker D8 Discover system (Asset Tag V012842) equipped with asealed tube source and a Hi-Star area detector (Bruker AXS, Madison,Wis.). The X-Ray generator is operated at a voltage of 40 kV and acurrent of 35 mA. The powder sample is placed in a nickel holder. Twoframes are registered with an exposure time of 120 s each. The data issubsequently integrated over the range of 4.5°-39° 2 theta with a stepsize of 0.02° and merged into one continuous pattern.

FIG. 1d shows the X-ray powder diffractogram of the sample which ischaracteristic of crystalline drug substance.

Representative XRPD peaks from Compound I-2.HCl.H₂O

XRPD Angle Peaks (2-Theta ± 0.2) Intensity % *1 6.6 100.0 *2 19.5 46.5 316.8 37.8 4 22.9 36.0 5 13.9 27.0 6 7.3 23.4 7 13.0 22.7 8 16.5 21.2 *924.7 20.9 10 17.7 20.8 11 31.1 19.6 12 15.8 19.3 *13 8.1 18.5 14 17.118.4 15 12.7 17.2 16 16.0 17.2 17 14.5 16.5 18 20.6 16.0 19 32.7 15.5*20 11.2 15.2 21 33.9 11.3

Thermo Analysis of Compound I-2.HCl.H₂O

Thermogravimetric analysis (TGA) for Compound I-2.HCl.H₂O was performedon the TA Instruments TGA model Q5000. Compound I-2.HCl.H₂O was placedin a platinum sample pan and heated at 10° C./min to 400° C. from roomtemperature. The thermogram (FIG. 2d ) demonstrates a weight loss of2.9% from room temperature to 100° C., and a weight loss of 0.6% from100° C. to 222° C., which is consistent with theoretical monohydrate(3.5%).

Differential Scanning Calorimetry of Compound I-2.HCl.H₂O

A DSC thermogram for Compound I-2.HCl.H₂O was obtained using TAInstruments DSC Q2000. Compound I-2.HCl.H₂O was heated at 2° C./min to275° C. from −20° C., and modulated at ±1° C. every 60 sec. The DSCthermogram (FIG. 3d ) reveals an endothermic peak below 200° C., whichcould corresponds to the loss of 1 equivalent of HCl.Melting/recrystallization occurs between 215-245° C., followed bydegradation.

Example 14 Compound I-2.HCl.2H₂O

Compound I-2.HCl.2H₂O can be formed from Compound I-2.2 HCl. (E29244-17)A suspension of Compound I-2.2 HCl (10.0 g, 18.6 mmol) in isopropylalcohol (40 mL) and water (10 mL) is warmed at 50° C. for about 1 h andthen cooled to below 10° C. The solid is collected by filtration. Thefilter-cake is washed with 80/20 isopropyl alcohol/water (2×10 mL) andair-dried to afford Compound I-2.HCl.2H₂O as a yellow powder.

XRPD of Compound I-2.HCl.2H₂O

The powder x-ray diffraction measurements were performed usingPANalytical's X-pert Pro diffractometer at room temperature with copperradiation (1.54060° A). The incident beam optic was comprised of avariable divergence slit to ensure a constant illuminated length on thesample and on the diffracted beam side, a fast linear solid statedetector was used with an active length of 2.12 degrees 2 theta measuredin a scanning mode. The powder sample was packed on the indented area ofa zero background silicon holder and spinning was performed to achievebetter statistics. A symmetrical scan was measured from 4-40 degrees 2theta with a step size of 0.017 degrees and a scan step time of 15.5 s.

FIG. 1d shows the X-ray powder diffractogram of the sample which ischaracteristic of crystalline drug substance.

Representative XRPD peaks from Compound I-2.HCl.2H₂O

XRPD Angle (2- Peaks Theta ± 0.2) Intensity % 1 16.2 100.0 2 13.4 82.0*3 26.6 69.2 4 15.9 66.3 5 15.5 63.8 6 17.1 55.3 7 28.3 55.3 8 7.2 51.89 20.7 49.7 10 15.3 46.2 11 20.2 44.6 12 28.0 41.2 13 19.9 40.4 14 17.639.1 15 26.3 39.0 *16 7.6 36.1 17 27.3 33.6 18 25.6 33.5 19 18.8 32.2 2027.0 29.1 21 20.8 28.7 22 22.5 28.0 23 13.0 23.8 *24 6.3 22.6 25 25.222.6 26 14.3 22.4 27 19.1 20.8 28 25.1 19.7 29 13.7 19.0 30 14.0 17.4 3133.0 16.2 *32 23.3 15.7 33 16.6 15.1 34 29.6 14.9 35 29.9 14.8 36 27.614.8 37 32.1 13.3 *38 24.6 13.1 39 30.8 11.1

Thermo Analysis of Compound I-2.HCl.2H₂O

The TGA (Thermogravimetric Analysis) thermographs were obtained using aTA instrument TGA Q500 respectively at a scan rate of 10° C./min over atemperature range of 25-300° C. For TGA analysis, samples were placed inan open pan. The thermogram demonstrates a weight loss of ˜6 from roomtemperature to 100° C., which is consistent with theoretical dihydrate(6.7%).

Differential Scanning Calorimetry of Compound I-2.HCl.2H₂O

A DSC (Differential Scanning calorimetry) thermographs were obtainedusing a TA instruments DSC Q2000 at a scan rate of 10° C./min over atemperature range of 25-300° C. For DSC analysis, samples were weighedinto aluminum hermetic T-zero pans that were sealed and punctured with asingle hole. The DSC thermogram reveals dehydration between roomtemperature and 120° C. followed by melting/recrystallization between170-250° C.

Crystal Structure of Compound I-2.HCl with Water

180 mg Compound I-2.HCl was added to a vial with 0.8 mL 2-propanol and0.2 mL water. The sealed vial was kept in an oven at 70° C. for twoweeks. Diffraction quality crystals were observed.

A yellow needle shape crystal with dimensions of 0.15×0.02×0.02 mm³ wasselected, mounted on a MicroMount and centered on a Bruker APEX IIdiffractometer (V011510). Then a kapton tube with water inside coveredthe pin. The tube was sealed to make sure the crystal is equilibratedwith water for two days before the diffraction experiments. Threebatches of 40 frames separated in reciprocal space were obtained toprovide an orientation matrix and initial cell parameters. Final cellparameters were collected and refined was completed based on the fulldata set.

A diffraction data set of reciprocal space was obtained to a resolutionof 106° 2θ angle using 0.5° steps with exposure times 20 s each framefor low angle frames and 60 s each frame for high angle frames. Datawere collected at room temperature. Integration of intensities andrefinement of cell parameters were conducted using the APEXII software.

Crystal Data

C₂₄H₂₈ClN₅O₄S M_(r) = 518.02 Monoclinic, P2₁/n a = 5.4324 (5) Å b =35.483 (4) Å c = 13.3478 (12) Å β = 100.812 (5)° V = 2527.2 (4) Å³

Example 15 Cellular ATR Inhibition Assay

Compounds can be screened for their ability to inhibit intracellular ATRusing an immunofluorescence microscopy assay to detect phosphorylationof the ATR substrate histone H2AX in hydroxyurea treated cells. HT29cells are plated at 14,000 cells per well in 96-well black imagingplates (BD 353219) in McCoy's 5A media (Sigma M8403) supplemented with10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycinsolution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (SigmaG7513), and allowed to adhere overnight at 37° C. in 5% CO₂. Compoundsare then added to the cell media from a final concentration of 25 μM in3-fold serial dilutions and the cells are incubated at 37° C. in 5% CO₂.After 15min, hydroxyurea (Sigma H8627) is added to a final concentrationof 2 mM.

After 45 min of treatment with hydroxyurea, the cells are washed in PBS,fixed for 10 min in 4% formaldehyde diluted in PBS (Polysciences Inc18814), washed in 0.2% Tween-20 in PBS (wash buffer), and permeabilisedfor 10 min in 0.5% Triton X-100 in PBS, all at room temperature. Thecells are then washed once in wash buffer and blocked for 30 min at roomtemperature in 10% goat serum (Sigma G9023) diluted in wash buffer(block buffer). To detect H2AX phosphorylation levels, the cells arethen incubated for 1 h at room temperature in primary antibody (mousemonoclonal anti-phosphorylated histone H2AX Ser139 antibody; Upstate05-636) diluted 1:250 in block buffer. The cells are then washed fivetimes in wash buffer before incubation for 1 h at room temperature inthe dark in a mixture of secondary antibody (goat anti-mouse Alexa Fluor488 conjugated antibody; Invitrogen A11029) and Hoechst stain(Invitrogen H3570); diluted 1:500 and 1:5000, respectively, in washbuffer. The cells are then washed five times in wash buffer and finally100 ul PBS is added to each well before imaging.

Cells are imaged for Alexa Fluor 488 and Hoechst intensity using the BDPathway 855 Bioimager and Attovision software (BD Biosciences, Version1.6/855) to quantify phosphorylated H2AX Ser139 and DNA staining,respectively. The percentage of phosphorylated H2AX-positive nuclei in amontage of 9 images at 20× magnification is then calculated for eachwell using BD Image Data Explorer software (BD Biosciences Version2.2.15). Phosphorylated H2AX-positive nuclei are defined asHoechst-positive regions of interest containing Alexa Fluor 488intensity at 1.75-fold the average Alexa Fluor 488 intensity in cellsnot treated with hydroxyurea. The percentage of H2AX positive nuclei isfinally plotted against concentration for each compound and IC50 s forintracellular ATR inhibition are determined using Prism software(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, SanDiego Calif., USA).

The compounds described herein can also be tested according to othermethods known in the art (see Sarkaria et al, “Inhibition of ATM and ATRKinase Activities by the Radiosensitizing Agent, Caffeine: CancerResearch 59: 4375-5382 (1999); Hickson et al, “Identification andCharacterization of a Novel and Specific Inhibitor of theAtaxia-Telangiectasia Mutated Kinase ATM” Cancer Research 64: 9152-9159(2004); Kim et al, “Substrate Specificities and Identification ofPutative Substrates of ATM Kinase Family Members” The Journal ofBiological Chemistry, 274(53): 37538-37543 (1999); and Chiang et al,“Determination of the catalytic activities of mTOR and other members ofthe phosphoinositide-3-kinase-related kinase family” Methods Mol. Biol.281:125-41 (2004)).

Example 16 ATR Inhibition Assay

Compounds can be screened for their ability to inhibit ATR kinase usinga radioactive-phosphate incorporation assay. Assays are carried out in amixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl₂ and 1 mM DTT. Finalsubstrate concentrations are 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP,Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).

Assays are carried out at 25° C. in the presence of 5 nM full-lengthATR. An assay stock buffer solution is prepared containing all of thereagents listed above, with the exception of ATP and the test compoundof interest. 13.5 μL of the stock solution is placed in a 96 well platefollowed by addition of 2 μL of DMSO stock containing serial dilutionsof the test compound (typically starting from a final concentration of15 μM with 3-fold serial dilutions) in duplicate (final DMSOconcentration 7%). The plate is pre-incubated for 10 minutes at 25° C.and the reaction initiated by addition of 15 μL [γ-33P]ATP (finalconcentration 10 μM).

The reaction is stopped after 24 hours by the addition of 30 μL 0.1Mphosphoric acid containing 2 mM ATP. A multiscreen phosphocellulosefilter 96-well plate (Millipore, Cat no. MAPHNOB50) is pretreated with100 μL 0.2M phosphoric acid prior to the addition of 450 μL of thestopped assay mixture. The plate is washed with 5×200 μL 0.2M phosphoricacid. After drying, 100 μL Optiphase ‘SuperMix’ liquid scintillationcocktail (Perkin Elmer) is added to the well prior to scintillationcounting (1450 Microbeta Liquid Scintillation Counter, Wallac).

After removing mean background values for all of the data points,Ki(app) data are calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego Calif., USA).

In general, the compounds of the present invention are effective forinhibiting ATR. Compounds I-1, I-2, II-1, II-2, II-3 and II-4 inhibitATR at Ki values below 0.001 μM.

Example 17 Cisplatin Sensitization Assay

Compounds can be screened for their ability to sensitize HCT116colorectal cancer cells to Cisplatin using a 96 h cell viability (MTS)assay. HCT116 cells, which possess a defect in ATM signaling toCisplatin (see, Kim et al.; Oncogene 21:3864 (2002); see also, Takemuraet al.; JBC 281:30814 (2006)) are plated at 470 cells per well in96-well polystyrene plates (Costar 3596) in 150 μ1 of McCoy's 5A media(Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539),and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at37° C. in 5% CO₂. Compounds and Cisplatin are then both addedsimultaneously to the cell media in 2-fold serial dilutions from a topfinal concentration of 10 μM as a full matrix of concentrations in afinal cell volume of 200 μl, and the cells are then incubated at 37° C.in 5% CO₂. After 96 h, 40 μl of MTS reagent (Promega G358a) is added toeach well and the cells are incubated for 1 h at 37° C. in 5% CO₂.Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384reader (Molecular Devices) and the concentration of compound required toreduce the IC50 of Cisplatin alone by at least 3-fold (to 1 decimalplace) can be reported.

Example 18 Single Agent HCT116 Activity

Compounds can be screened for single agent activity against HCT116colorectal cancer cells using a 96 h cell viability (MTS) assay. HCT116are plated at 470 cells per well in 96-well polystyrene plates (Costar3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10%foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycinsolution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (SigmaG7513), and allowed to adhere overnight at 37° C. in 5% CO₂. Compoundsare then added to the cell media in 2-fold serial dilutions from a topfinal concentration of 10 μM as a full matrix of concentrations in afinal cell volume of 200 μl, and the cells are then incubated at 37° C.in 5% CO₂. After 96 h, 40 μl of MTS reagent (Promega G358a) is added toeach well and the cells are incubated for 1h at 37° C. in 5% CO₂.Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384reader (Molecular Devices) and IC50 values can be calculated.

Data for Examples 18-21

Single agent Cisplatin Cmpd HT116 ATR inhibition ATR cellularsensitization No. IC50 (nM) Ki (nM) IC50 (nM) (nM) II-1 62 <1 18 39 II-246 <1 — 29 II-3 66 0.148 10 39 II-4 — 0.2 — —

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds, methods, and processes of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments that have been represented by way of exampleherein.

1-82. (canceled)
 83. A compound of formula II:

or a pharmaceutically acceptable salt thereof, wherein each R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), R^(9b), R¹⁰, R¹¹, and R¹² is independently hydrogen or deuterium, and at least one of R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), R^(3c), R⁴, R⁵, R⁶, R⁷, R⁸, R^(9a), R^(9b), R¹⁰, R¹¹, and R¹² is deuterium. 84-125. (canceled)
 126. The compound of claim 83, wherein each of R^(1a), R^(1b), and R^(1c) is deuterium.
 127. The compound of claim 83, wherein R² is deuterium.
 128. The compound of claim 83, wherein each of R^(3a), R^(3b), and R^(3c) is deuterium.
 129. The compound of claim 83, wherein each of R^(1a), R^(1b), R^(1c), R^(3a), R^(3b), and R^(3c) is deuterium.
 130. The compound of claim 83, wherein each of R^(1a), R^(1b), R^(1c), R², R^(3a), R^(3b), and R^(3c) is deuterium.
 131. The compound of claim 83, wherein at least one of R^(9a) and R^(9b) is deuterium.
 132. The compound of claim 83, wherein each of R^(9a) and R^(9b) is deuterium.
 133. The compound of claim 83, wherein at least one of R¹⁰, R¹¹, and R¹² is deuterium.
 134. The compound of claim 83, wherein at least two of R¹⁰, R¹¹, and R¹² is deuterium.
 135. The compound of claim 83, wherein each of R¹⁰, R¹¹, and R¹² is deuterium.
 136. The compound of claim 83, wherein each of R^(9a), R^(9b), R¹⁰, R¹¹, and R¹² is deuterium.
 137. The compound of claim 83, wherein at least one of R⁴, R⁵, R⁶, R⁷, and R⁸ is deuterium.
 138. The compound of claim 131, wherein at least one of R⁴, R⁵, R⁶, R⁷, and R⁸ is deuterium.
 139. The compound of claim 133, wherein at least one of R⁴, R⁵, R⁶, R⁷, and R⁸ is deuterium.
 140. The compound of claim 131, wherein R² is deuterium.
 141. The compound of claim 133, wherein R² is deuterium.
 142. The compound of claim 83, selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 143. The compound of claim 142, selected from the group consisting of compound II-1, II-2, II-3, and II-4.
 144. A pharmaceutical composition comprising a compound of claim 83 and a pharmaceutically acceptable carrier. 